Antibodies which bind human cxcr3

ABSTRACT

Antibodies and antigen-binding fragments of antibodies that bind human CXCR3 are disclosed. In preferred embodiments, the antibodies are human. Nucleic acids and vectors encoding the antibodies or portions thereof, recombinant cells that contain the nucleic acids, and compositions comprising the antibodies or antigen-binding fragments are also disclosed. The invention also provides therapeutic and diagnostic methods which employ the antibodies and antigen-binding fragments.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/170,233, filed Jun. 28, 2011, which is a Divisional of U.S. patentapplication Ser. No. 12/214,891, filed Jun. 23, 2008, now U.S. Pat. No.7,994,287, which is a Divisional of U.S. patent application Ser. No.10/949,135, filed Sep. 24, 2004, now U.S. Pat. No. 7,405,275, whichclaims the benefit of U.S. Provisional Application No. 60/505,697, filedSep. 24, 2003. The entire contents of each of the foregoing applicationsare incorporated herein by this reference.

BACKGROUND OF THE INVENTION

C—X—C chemokine receptor-3 (CXCR3) is expressed on certain leukocytes,such as activated T cells and NK cells. The CXCR3 receptor bindsligands, such as Interferon Gamma-inducible 10 kD Protein (IP-10),Monokine Induced by Gamma interferon (MIG; Mig), Interferon-inducibleT-cell Alpha Chemoattractant (I-TAC) and B cell-attracting chemokine-1(BCA-1). Certain forms of CXCR3 also bind platelet factor-4 (PF-4,Lasagni et al. (2003) J. Exp. Med. 197:1537-1549). The expression ofsome of the CXCR3 ligands (IP-10, MIG and I-TAC), is induced in tissuesby inteferons or Tumor Necrosis Factor (TNF), potent mediators ofinflammation (Farber, J. M. (1997) J Leukoc. Biol. 61:246-257; Piali, etal. (1998) Eur. J. Immunol. 28:961-972; Cole et al. (1998) J. Exp. Med.187:2009-2021). Because of these findings, it has been postulated thatduring inflammation, expression of ligands for CXCR3 is upregulated,resulting in recruitment of CXCR3+ lymphocytes into the inflamed tissue.The infiltrating CXCR3+ lymphocytes can contribute to adversepathological effects of inflammation. Inhibiting the activities ofCXCR3, therefore, can have beneficial anti-inflammatory effects. Thereis a need for therapeutic agents that inhibit CXCR3 function.

SUMMARY OF THE INVENTION

The invention relates to antibodies and antigen-binding fragments ofantibodies, such as human monoclonal antibodies, which bind human CXCR3.In certain embodiments, the antibodies and antigen-binding fragments canalso bind ligand-binding variants of human CXCR3 and/or fragments ofhuman CXCR3. In one embodiment, the antibody or antigen-binding fragmentthereof binds an extracellular loop of human CXCR3. In anotherembodiment, the antibody or antigen-binding fragment thereof inhibitsthe binding of a ligand (e.g., IP-10, MIG or I-TAC, or a CXCR3-bindingvariant or fragment of IP-10, MIG or I-TAC) to human CXCR3. In otherembodiments, the antibody or antigen-binding fragment inhibits bindingof at least 3 ligands, such as IP-10, MIG and I-TAC, to human CXCR3. Inother embodiments, the antibody or antigen-binding fragment inhibitsligand-induced activity of CXCR3. For example, an antibody of theinvention can inhibit CXCR3-mediated signal transduction, intracellularcalcium (Ca++) release, more specifically the induction of a rapid andtransient increase in the concentration of cytosolic free calcium[Ca²⁺]_(i) (calcium flux), chemotaxis, cell differentiation or cellproliferation that is induced upon ligand binding. In particularembodiments, the antibody or antigen-binding fragment competitivelyinhibits binding of human mAb 5H7 or human mAb 7H5 to CXCR3, or has theepitopic specificity of human mAb 5H7 or human mAb 7H5.

In some embodiments, the antibody or antigen-binding fragment comprisesone, two or three heavy chain complementarity determining regions(HCDR1, HCDR2 and/or HCDR3) having the amino acid sequences of heavychain CDR1, CDR2 and CDR3 of human mAb 5H7. In certain embodiments, oneamino acid residue in the heavy chain CDR1, one or two amino acidresidues in the heavy chain CDR2, and/or one, two or three amino acidresidues in CDR3 can be conservatively substituted as described herein.The antibody or antigen-binding fragment can further comprise one, twoor three light chain complementarity determining regions (LCDR1, LCDR2and/or LCDR3) having the amino acid sequences of light chain CDR1, CDR2and CDR3 of human mAb 5H7. In certain embodiments, one or two amino acidresidues in the light chain CDR1, one amino acid residue in the lightchain CDR2 and/or one or two amino acid residues in the light chain CDR3can be conservatively substituted. Preferably, the antibody orantigen-binding fragment comprises the three heavy chain CDRs and thethree light chain CDRs of human mAb 5H7. In more particular embodiments,the antibody or antigen-binding fragment comprises the heavy chainvariable region of human mAb 5H7 (5H7 VH, SEQ ID NO: 2) and the lightchain variable region of human mAb 5H7 (5H7 VK, SEQ ID NO:10).

In other embodiments, the antibody or antigen-binding fragment thereofcomprises one, two or three heavy chain complementarity determiningregions (HCDR1, HCDR2 and/or HCDR3) having the amino acid sequences ofthe heavy chain CDR1, CDR2, and CDR3 of human mAb 7H5. In certainembodiments, one amino acid residue in the heavy chain CDR1, one or twoamino acid residues in the heavy chain CDR2 and/or one, two or threeamino acid residues in the heavy chain CDR3 can be conservativelysubstituted. The antibody or antigen-binding fragment can furthercomprise one, two or three light chain complementarity determiningregions (LCDR1, LCDR2 and/or LCDR3) having the amino acid sequences ofthe light chain CDR1, CDR2, and CDR3 of human mAb 7H5. In certainembodiments, one or two amino acid residues in the light chain CDR1, oneamino acid residue in the light chain CDR2 and/or one or two amino acidresidues in the light chain CDR3 can be conservatively substituted.Preferably, the antibody or antigen-binding fragment comprises the threeheavy chain CDRs and the three light chain CDRs of human mAb 7H5. Inmore particular embodiments, the antibody or antigen-binding fragmentcomprises the heavy chain variable region of human mAb 7H5 (7H5 VH, SEQID NO:18) and the light chain variable region of human mAb 7H5 (7115 VK,SEQ ID NO:26).

Preferred antibodies and antigen-binding fragments that bind human CXCR3include human antibodies, chimeric antibodies, humanized antibodies,single chain antibodies and antigen-binding fragments of the foregoing,such as Fab fragments, Fab′ fragments, F(ab ′)₂ fragments and Fvfragments. Particularly preferred antibodies and antigen-bindingfragments are human. In specific embodiments, the invention is human mAb5H7 or an antigen-binding fragment of human mAb 5H7 or human mAb 7H5 oran antigen-binding fragment of human mAb 7H5.

The invention also relates to the heavy chains, light chains andantigen-binding portions of the heavy chains and light chains of theantibodies described herein. The invention also relates to fusionproteins comprising an antibody or portion thereof (e.g., heavy chain,light chain, variable region) of the invention and a non-immunoglobulinmoiety. The invention also relates to immunoconjugates comprising anantibody or antigen-binding fragment of the invention and a secondmoiety, such as a toxin (e.g., cytotoxin, cytotoxic agent), atherapeutic agent (e.g., a chemotherapeutic agent, an antimetabolite, analkylating agent, an anthracycline, an antibiotic, an anti-mitoticagent, a biological response modifier (e.g., a cytokine, a growth factor(e.g., a neurotrophic factor)), a plasminogen activator, a radionuclide(e.g., a radioactive ion)), an enzyme or a detectable label, e.g., aradionuclide.

The invention also relates to isolated and/or recombinant nucleic acidsencoding the antibodies, antigen-binding fragments, heavy chains, lightchains and portions of the heavy chains and light chains of theantibodies described herein, or fusion proteins and to expressionconstructs or vectors comprising same. The invention also relates to anexpression construct comprising a recombinant nucleic acid molecule thatencodes a heavy chain or an antigen-binding portion thereof, and to anexpression construct comprising a recombinant nucleic acid molecule thatencodes a light chain or an antigen-binding portion thereof. Theinvention also relates to a host cell that comprises a nucleic acid ofthe invention. The invention also relates to an isolated cell whichproduces a heavy chain or an antigen-binding portion thereof, a lightchain or an antigen-binding portion thereof. In specific embodiments,the invention is hybridoma 5H7 or hybridoma 7H5.

The invention also relates to a composition comprising an antibody orantigen-binding fragment thereof, e.g., a monoclonal antibody (e.g. ahuman monoclonal antibody or antigen-binding fragment thereof, a heavychain or antigen-binding portion thereof, a light chain orantigen-binding portion thereof, a chimeric antibody or antigen-bindingfragment thereof), or an immuno-conjugate of an antibody describedherein and a physiologically acceptable carrier. In specificembodiments, the composition comprises human monoclonal antibody 5H7and/or human monoclonal antibody 7H5 and a physiologically acceptablecarrier.

The invention also relates to a method of treating a subject having aninflammatory disease or disorder comprising administering to saidsubject an effective amount of an antibody or antigen-binding fragmentof the invention. In particular embodiments, the subject is a human. Inother particular embodiments, the subject has an inflammatory boweldisease, such as ulcerative colitis or Crohn's disease, an autoimmunedisease, such as rheumatoid arthritis or multiple sclerosis.

The invention further relates to an antibody, antigen-binding fragmentof an antibody (including antigen-binding fragments), immunoglobulinchain (including antigen-binding portions), fusion protein orimmuno-conjugate as described herein for use in therapy (includingprophylaxis) or diagnosis, and to the use of an antibody,antigen-binding fragment of an antibody, fusion protein orimmuno-conjugate of the invention for the manufacture of a medicamentfor the treatment of a particular disease or condition as describedherein. For example, the disease or condition can be a disease orcondition mediated by a cell expressing CXCR3 (e.g., a T helper-1 cell,an eosinophil), an inflammatory disease (e.g., inflammatory boweldisease (e.g., ulcerative colitis, Crohn's disease)), an autoimmunedisease (e.g., rheumatoid arthritis, multiple sclerosis, Graves'disease, diabetes), cancer (e.g., a lymphoproliferative disease), or aninflammatory disease precipitated by foreign matter (e.g., graftrejection, response to bacterial or viral infection), a respiratoryinflammatory disease (e.g. chronic obstructive pulmonary disease)).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the mean fluorescence (MF) intensity displayedby the binding of human monoclonal antibody (mAb) 5H7, human monoclonalantibody 7H5 or murine monoclonal antibody 1C6 to CXCR3 transfectants.The 100% value of mean fluorescence intensity, from which the EC50 wascalculated, is the intensity of binding by 10 μg/ml of each antibody.The EC50 values are 0.5 nM, 1.2 nM and 2.3 nM for human mAb 5H7, humanmAb 7H5 and murine mAb 1C6, respectively.

FIG. 2 is a histogram depicting the number of CXCR3 transfectantsMigrating in an in vitro chemotaxis assay toward a ligand (IP-10, MIG orI-TAC) in the absence (“positive”) or presence of a monoclonal antibodyto human CXCR3 (murine monoclonal antibody 1C6, human monoclonalantibody 5H7, subclone 4, elution tubes (1) or (2) or human monoclonalantibody 5H7, subclone 6, elution tubes (1) or (2)). The results showthat human monoclonal antibody 5H7 inhibits CXCR3-mediated chemotaxis inresponse to IP-10, MIG and I-TAC.

FIG. 3 is a graph showing the percent inhibition of chemotaxis of CXCR3transfectants toward IP-10, MIG or I-TAC by human monoclonal antibody5H7 in an in vitro chemotaxis assay. The IC50 of human mAb 5H7 againstIP-10 is 0.7 μg/ml, against MIG is 2.9 μg/ml and against I-TAC is 1.2μg/ml.

FIG. 4 is a graph showing the percent inhibition of chemotaxis of CXCR3transfectants toward IP-10, MIG or I-TAC by murine monoclonal antibody1C6 in an in vitro chemotaxis assay. The IC50 of murine mAb 1C6 againstIP-10 is 0.8 μg/ml, against MIG is 93 μg/ml and against I-TAC is 23.9μg/ml.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “functionally rearranged” refers to a segmentof DNA from an immunoglobulin locus which has undergone V(D)Jrecombination, with or without insertion or deletion of nucleotide(s)(e.g., N nucleotides, P nucleotides) and/or somatic mutation, therebyproducing an immunoglobulin gene which encodes an immunoglobulinvariable region or immunoglobulin chain (e.g., heavy chain, lightchain). A functionally rearranged immunoglobulin gene can be directly orindirectly identified using suitable methods, such as, for example,nucleotide sequencing, hybridization (e.g., Southern blotting, Northernblotting) using probes which can anneal to coding joints between genesegments (e.g., VH, VL, D, JH, JL) or enzymatic amplification ofimmunoglobulin genes (e.g., polymerase chain reaction) with primerswhich can anneal to coding joints between gene segments. Whether a cellproduces an antibody comprising a particular variable region or avariable region comprising a particular sequence (e.g., a CDR sequence)can also be determined using suitable methods. In one example, mRNA canbe isolated from an antibody producing cell (e.g., a hybridoma) and usedto produce cDNA. The cDNA can be cloned and sequenced or can beamplified (e.g., by polymerase chain reaction) using a first primerwhich anneals specifically to a portion of the variable region ofinterest (e.g., CDR, coding joint) and a second primer which annealsspecifically to non-variable region sequences (e.g., C_(H)1, C_(L)).

As used herein, the phrase “of human origin” refers to antibodies,antigen-binding fragments of antibodies and portions or regions ofantibodies (e.g., variable regions, complementarity determining regions(CDRs), framework regions (FRs), constant regions) having amino acidsequences that are encoded by nucleotide sequences derived from human(Homo sapiens) germ line immunoglobulin genes. For example, an antibodyof human origin can be encoded by human germ line immunoglobulin genesthat have been functionally rearranged to produce a functional gene thatcan be expressed to produce an antibody. As described herein,functionally rearranged genes that encode an antibody chain can includesequences that are not found in the germ line, such as N nucleotides andP nucleotides, and mutations that can occur as part of the processesthat produce high-affinity antibodies (e.g., somatic mutation, affinitymaturation, clonal selection). Functionally rearranged immunoglobulingenes of human origin, including those that include non-germ linesequences, can be generated via natural processes in a suitable in vivoexpression system (e.g., a human, a human-antibody transgenic animal),artificially using any suitable methods (e.g., recombinant DNAtechnology, phage display) or any combination of natural and artificialprocesses. Antibodies, antigen-binding fragments of antibodies andportions or regions of antibodies of human origin can be produced, forexample, by expression of a nucleic acid of non-human origin (e.g., asynthetic nucleic acid) that has the requisite nucleotide sequence.

An antibody, antigen-binding fragment of an antibody or a portion of anantibody (e.g., a framework region) “of human origin” can have an aminoacid sequence that is encoded by a nucleic acid that has a nucleotidesequence that is a consensus of the nucleotide sequences of a number ofnaturally occurring human antibody genes or human germ line sequences,or have an amino acid sequence that is a consensus of the amino acidsequences of a number of naturally occurring human antibodies or aminoacid sequences encoded in the human germ line. A number of humanantibody consensus sequences are available, including consensussequences for the different subgroups of human variable regions (see,Kabat, E. A., et al., Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, U.S.Government Printing Office (1991)). The Kabat database and itsapplications are freely available on line, e.g. via IgBLAST at theNational Center for Biotechnology Information, Bethesda, Md. (also see,Johnson, G. and Wu, T. T., Nucleic Acids Research 29:205-206 (2001)).

As used herein, the phrase “human antibody” refers to an antibody or anantigen-binding fragment of an antibody in which the variable andconstant regions (if present) have amino acid sequences that are encodedby nucleotide sequences derived from human (Homo sapiens) germlineimmunoglobulin genes. A “human antibody” can include sequences that arenot encoded in the germline (e.g., due to N nucleotides, P nucleotides,and mutations that can occur as part of the processes that producehigh-affinity antibodies such as, somatic mutation, affinity maturation,clonal selection) that occur as a result of biological processes in asuitable in vivo expression system (e.g., a human, a human-antibodytransgenic animal). Antibodies, antigen-binding fragments of antibodiesand portions or regions of human antibodies can be produced, forexample, by expression of a nucleic acid of non-human origin (e.g., asynthetic nucleic acid) that has the requisite nucleotide sequence.

As used herein, the phrase “CDR-grafted” antibody refers to an antibodyor an antigen-binding fragment of an antibody which comprises a CDR thatis not naturally associated with the framework regions of the antibodyor antigen-binding fragment. Generally the CDR is from an antibody froma first species and the framework regions and constant regions (ifpresent) are from an antibody from a different species. The CDR-graftedantibody can be a “humanized antibody.”

As used herein, “humanized antibody” refers to an antibody orantigen-binding fragment thereof comprising a CDR that is not of humanorigin and framework and/or constant regions that are of human origin.For example, a humanized antibody can comprise a CDR derived from anantibody of nonhuman origin (e.g., natural antibody such as a murine(e.g., mouse, rat) antibody, artificial antibody) that binds a humanCXCR3, and framework and constant regions (if present) of human origin(e.g., a human framework region, a human consensus framework region, ahuman constant region (e.g., CL, CH1, hinge, CH2, CH3, CH4)).CDR-grafted single chain antibodies containing a CDR of non-human originand framework and constant regions (if present) of human origin (e.g.,CDR-grafted scFV) are also encompassed by the term humanized antibody.

As used herein, the term “chimeric antibody” refers to an antibody orantigen-binding fragment thereof comprising a variable region from anantibody from a first species and a constant region from an antibodyfrom a different species. None of the portions which comprise a chimericantibody needs to be of human origin. For example, a chimeric antibodycan comprise a variable region from a rodent (e.g., mouse) antibody anda constant region of a non-human primate antibody (e.g., a chimpanzeeconstant region).

The antibody of the invention can be a single chain antibody (e.g., asingle chain Fv (scFv)) and can include a linker moiety (e.g., a linkerpeptide) not found in native antibodies. For example, a scFv cancomprise a linker peptide, such as two to about twenty glycine residuesor other suitable linker, which connects a heavy chain variable regionto a light chain variable region. For the purposes of the invention, thepresence of such a linker does not affect the status of the single chainantibody as being “of human origin” or “human.” For example, a humanscFv can comprise a human heavy chain variable region and a human lightchain variable region which are connected through a suitable peptidelinker.

As used herein, “conservative amino acid substitution” refers to thereplacement of one amino acid by another within the following groups:Ala, Val, Leu, and Ile; Ser and Thr; Asp and Glu; Asn and Gln; Lys andArg; Phe and Tyr.

As used herein, a “CXCR3 protein” or “human CXCR3” refers to naturallyoccurring or endogenous human CXCR3 proteins and to proteins having anamino acid sequence which is the same as that of a naturally occurringor endogenous human CXCR3 protein (e.g., recombinant proteins).Accordingly, as defined herein, “CXCR3 protein” or “human CXCR3”includes mature protein, polymorphic or allelic variants, and otherisoforms of human CXCR3 (e.g., produced by alternative splicing or othercellular processes), and modified or unmodified forms of the foregoing(e.g., glycosylated, unglycosylated, phosphorylated or unphosphorylatedCXCR3 proteins). Naturally occurring or endogenous human CXCR3 proteinsinclude wild type proteins such as mature CXCR3, polymorphic or allelicvariants and other isoforms that occur naturally in humans. A nucleotidesequence encoding a full length human (Homo sapiens) CXCR3, used in thestudies described herein is disclosed as “SEQ ID NO:1” in U.S. Pat. No.6,184,358 B1, the contents of which are incorporated herein by referencein their entirety. This sequence is presented herein as SEQ ID NO: 33.The sequence of a human CXCR3 also is available in GenBank underaccession number NM_(—)001504. The entire teachings of GenBank AccessionNumber NM_(—)001504 also are incorporated herein by reference. Thenucleotide sequence of SEQ ID NO:33 has an open-reading frame fromnucleotides 69-1175 (including stop codon). The amino acid sequence of ahuman CXCR3 encoded by SEQ ID NO: 33 is presented as SEQ ID NO: 34 (“SEQID NO:2” of U.S. Pat. No. 6,184,358 B1).

Human CXCR3 is a G protein-coupled receptor with an N-terminalextracellular region, seven transmembrane domains, three intracellularloops, three extracellular loops and a C-terminal cytoplasmic region.Ligand-binding variants of human CXCR3 can comprise an N-terminalextracellular region, at least one, two, three, four, five, six,preferably seven transmembrane domains, at least one, two, preferablythree intracellular loops, at least one, two, preferably threeextracellular loops and/or a C-terminal cytoplasmic region. TheN-terminal extracellular region of human CXCR3 is located at about aminoacid residues 1-58 of SEQ ID NO: 34, can be important for ligand bindingand can include the epitope recognized by murine monoclonal antibody 1C6previously described in U.S. Pat. No. 6,184,358 B1 and Qin et al. (1998)J. Clin. Invest. 101:746-754. The transmembrane domains are located atabout amino acid residues 59-79, 91-111, 127-147, 170-190, 224-244,257-277 and 302-322 of SEQ ID NO: 34. The extracellular loops of humanCXCR3 are located at about amino acid residues 112-126, 191-223, and278-301 of SEQ ID NO:34 and can be important for ligand binding. Forexample, the second extracellular loop (about residues 191-223 of SEQ IDNO:34) has been shown to be involved in ligand binding. Theintracellular loops of human CXCR3 are located at about amino acidresidues 80-90, 148-169, and 245-256 of SEQ ID NO:34 and, together withthe C-terminal cytoplasmic region at about amino acid residues 323-368of SEQ ID NO:34 can be important for function mediated by CXCR3 andinduced by ligand binding, e.g. signal transduction, intracellularcalcium release, chemotaxis, cell differentiation, cell proliferationand cell activation.

Allelic variants of human CXCR3 include both functional andnon-functional proteins. Functional allelic variants are naturallyoccurring amino acid sequence variants of the CXCR3 protein thatmaintain the ability to bind a CXCR3 ligand and/or perform aCXCR3-mediated function. Non-functional allelic variants arenaturally-occurring amino acid sequence variants of human CXCR3 proteinthat do not have the ability to bind a CXCR3 ligand and/or perform aCXCR3-mediated function. A number of allelic variants of human CXCR3have been identified and these, among others, are encompassed by theterm allelic variant. (See, e.g., Kato et al. (2000) Genes Immun.1:330-337.)

Ligand-binding variants and fragments of human CXCR3 comprise at leastone extracellular portion of human CXCR3. A ligand-binding variant orfragment of human CXCR3 can bind at least one ligand of CXCR3, such asIP-10, MIG, I-TAC, BCA-1 or a receptor-binding fragment of any of theforegoing. A ligand-binding variant or fragment of human CXCR3 caninclude a chemically synthesized peptide of CXCR3. A protein comprisingan extracellular portion of human CXCR3 can be, for example, a deletionmutant. Such a deletion mutant can be expressed from a recombinantnucleic acid molecule encoding at least that extracellular portion ofhuman CXCR3, for example. A library of nucleic acids encoding apopulation of ligand-binding CXCR3 fragments or variants can beexpressed to produce a diverse population of CXCR3 variants forscreening and/or subsequent selection of antibodies or antigen-bindingfragments which bind CXCR3 variants.

For example, a protein comprising an extracellular portion of humanCXCR3 can include at least a portion of the N-terminus of a human CXCR3,e.g., amino acid residues 1-37 or 1-58 of SEQ ID NO:34 and/or at leastthe second extracellular loop of a human CXCR3, e.g., amino acidresidues 191-223 of SEQ ID NO:34. A protein comprising an extracellularportion of a human CXCR3 can be a fusion protein having anextracellular, e.g., ligand-binding, portion of a human CXCR3 fused toanother molecule, e.g., a portion of another G protein-coupled receptor,a molecule having an entity which anchors the CXCR3 extracellularportion to a cell membrane, e.g., a heterologous transmembrane domain ora glycosylphosphatidylinositol membrane anchor, or a molecule designedto transduce a detectable signal when bound to a CXCR3 ligand.

“Human IP-10” includes immature and mature forms of human IP-10, e.g.after removal of the signal peptide (e.g. removal of residues 1-21 toleave residues 22-98 of SEQ ID NO:35 as the mature form). An amino acidsequence of a human (Homo sapiens) IP-10 used in the studies describedherein and deposited in GenPept under accession number NP_(—)001556 ispresented as SEQ ID NO:35. The entire teachings of GenPept AccessionNumber NP_(—)001556 are incorporated herein by reference. Areceptor-binding fragment of human IP-10 can comprise fewer residuesthan the mature form, e.g., comprise at least about 5, 6, 7, 10, 15, 20or more residues of SEQ ID NO:35. For example, receptor-bindingfragments of human IP-10 include the N-terminus (about residues 22-29 ofSEQ ID NO:35), the N-loop (about residues 33-38 of SEQ ID NO:35), the40s-loop and the 30s-loop (see, e.g., Booth et al. (2002) Biochemistry41:10418-10425).

“Human MIG” includes immature and mature forms of human MIG, e.g. afterremoval of the signal peptide (e.g. removal of residues 1-22 to leaveresidues 23-125 of SEQ ID NO:36 as the mature form). An amino acidsequence of a human (Homo sapiens) MIG used in the studies describedherein and deposited in GenPept under accession number NP_(—)002407 ispresented as SEQ ID NO:36. The entire teachings of GenPept AccessionNumber NP_(—)002407 are incorporated herein by reference. Areceptor-binding variant of human MIG can include active secretionvariants characterized by Liao et al. ((1995) J. Exp. Med.182:1301-1314). A receptor-binding fragment of human MIG can comprisefewer residues than the mature form, e.g., comprise at least about 5, 6,7, 10, 15, 20 or more residues of SEQ ID NO:36. For example, areceptor-binding fragment of human MIG can include the mature proteinN-terminus and/or the N-loop of MIG (e.g. about amino acid residues23-30 or 34-39 of SEQ ID NO:36, respectively). A receptor-bindingfragment of human MIG can include at least the C-terminus of MIG (aboutamino acids 96-125 of SEQ ID NO:36, Clark-Lewis et al. (2003) J. Biol.Chem. 278:289-295).

“Human I-TAC” includes immature and mature forms of human I-TAC, e.g.after removal of the signal peptide (e.g. removal of residues 1-21 toleave residues 22-94 of SEQ ID NO:37 as the mature form). An amino acidsequence of a human (Homo sapiens) I-TAC used in the studies describedherein and deposited in SwissProt under accession number O14625 ispresented as SEQ ID NO:37. The entire teachings of SwissProt AccessionNumber O14625 are incorporated herein by reference. A receptor-bindingfragment of human I-TAC can comprise fewer residues than the matureform, e.g., comprise at least about 5, 6, 7, 10, 15, 20 or more residuesof SEQ ID NO:37. For example, receptor-binding fragments of human I-TACinclude the mature protein N-terminus, the N-loop (e.g. about residues33-38 of SEQ ID NO:37), and mature protein N-terminal deletion mutants(e.g. I-TAC mutants having residues 23-94 or 24-94 of SEQ ID NO:37) (seee.g., Clark-Lewis et al. (2003) J. Biol. Chem. 278:289-295).

Antibodies and Antibody Producing Cells

In the past, murine mAbs against human CXCR3 have been generated butmost of them failed to completely inhibit binding of at least threeligands of CXCR3 tested, specifically IP-10, MIG and I-TAC. This may beexplained by studies that suggest that ligands bind to CXCR3 differentlyfrom each other (Cox et al. (2001) Mol. Pharmacol. 59:707-715). Incertain embodiments, antibodies (and cells which produce antibodies)that inhibit the binding of at least three ligands to human CXCR3 areprovided. As described herein, human mAb 5H7 and human mAb 7H5 whicheach bind human CXCR3 were produced. Studies using these mAbs revealedthat both human mAb 5H7 and human mAb 7H5 inhibited I-TAC inducedchemotaxis of cells that expressed human CXCR3 in an in vitro assay.Further studies revealed that both human mAb 5H7 and human mAb 7H5inhibited I-TAC-, IP-10- and MIG-induced chemotaxis of cells thatexpressed human CXCR3 in an in vitro assay.

The antibody of the invention can be polyclonal or monoclonal (mAb), andthe term “antibody” is intended to encompass both polyclonal andmonoclonal antibodies. The terms polyclonal and monoclonal refer to thedegree of homogeneity of an antibody preparation, and are not intendedto be limited to particular methods of production. The term “antibody”as used herein encompasses antigen-binding fragments of antibodies,including antigen-binding fragments of human, humanized, chimeric,CDR-grafted, veneered or single-chain antibodies and the like.

Antibodies which bind a CXCR3 protein can be selected from a suitablecollection of natural or artificial antibodies or raised against anappropriate immunogen in a suitable host. For example, antibodies can beraised by immunizing a suitable host (e.g., mouse, humanantibody-transgenic mouse, rat, rabbit, chicken, goat, non-human primate(e.g., monkey)) with a suitable immunogen, such as an isolated orpurified CXCR3 protein, a cell expressing a recombinant CXCR3 protein(e.g., a cell that expresses an exogenous nucleic acid encoding humanCXCR3 protein (CD183) (e.g. a CXCR3 transfectant)) or a chemicallysynthesized peptide of CXCR3, e.g. a peptide comprising at least 8, 9,10, 11, 12, 15, 20, 25, 30, 33, 35, 37, or 40 residues of anextracellular sequence of human CXCR3 (e.g., a peptide having amino acidresidues 1-37 or 191-223 of SEQ ID NO:2). In addition, cells expressinga recombinant CXCR3 protein, variant or ligand-binding fragment thereof,such as transfected cells, can be used in a screen for an antibody whichbinds thereto (See e.g., Chuntharapai et al., J. Immunol., 152:1783-1789 (1994); Chuntharapai et al., U.S. Pat. No. 5,440,021).

Preparation of the immunizing antigen, and polyclonal and monoclonalantibody production can be performed using any suitable technique. Avariety of methods have been described. (See, e.g., Kohler et al.,Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976);Milstein et al., Nature 266: 550-552 (1977); Koprowski et al., U.S. Pat.No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.);Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer'94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.),Chapter 11, (1991).) Generally, where a monoclonal antibody is desired,a hybridoma is produced by fusing a suitable cells from an immortal cellline (e.g., a myeloma cell line such as SP2/0, P3X63Ag8.653 or aheteromyeloma) with antibody-producing cells. Antibody-producing cellscan be obtained from the peripheral blood or, preferably the spleen orlymph nodes, of humans, human-antibody transgenic animals or othersuitable animals immunized with the antigen of interest. Cells thatproduce antibodies of human origin (e.g., a human antibody) can beproduced using suitable methods, for example, fusion of a humanantibody-producing cell and a heteromyeloma or trioma, orimmortalization of an activated human B cell via infection with EpsteinBarr virus. (See, e.g., U.S. Pat. No. 6,197,582 (Trakht); Niedbala etal., Hybridoma, 17:299-304 (1998); Zanella et al., J Immunol Methods,156:205-215 (1992); Gustafsson et al., Hum Antibodies Hybridomas,2:26-32 (1991).) The fused or immortalized antibody-producing cells(hybridomas) can be isolated using selective culture conditions, andcloned by limiting dilution. Cells which produce antibodies with thedesired specificity can be identified using a suitable assay (e.g.,ELISA).

Antibodies also can be prepared directly (e.g., synthesized or cloned)from an isolated antigen-specific antibody producing cell (e.g. a cellfrom the peripheral blood or, preferably the spleen or lymph nodesdetermined to produce an antibody with desired specificity), of humans,human-antibody transgenic animals or other suitable animals immunizedwith the antigen of interest (see, e.g. U.S. Pat. No. 5,627,052(Schrader)).

Other suitable methods of producing or isolating antibodies orantigen-binding fragments of the desired specificity can be used,including, for example, methods which select a recombinant antibody orantigen-binding fragment thereof from a library, such as a phage displaylibrary. Such libraries can contain antibodies or antigen-bindingfragments of antibodies that contain natural or artificial amino acidsequences. For example, the library can contain Fab fragments whichcontain artificial CDRs (e.g., random amino acid sequences) and humanframework regions. (See, for example, U.S. Pat. No. 6,300,064 (Knappik,et al.), the entire teachings of which are incorporated herein byreference.)

Human antibodies and nucleic acids encoding same can be obtained from ahuman or from human-antibody transgenic animals. Human-antibodytransgenic animals (e.g., mice) are animals that are capable ofproducing a repertoire of human antibodies, such as XENOMOUSE® (Abgenix,Fremont, Calif.), HUMAB-MOUSE®, KIRIN TC MOUSE® or KM-MOUSE® (MEDAREX®,Inc., Princeton, N.J.). Generally, the genome of human-antibodytransgenic animals has been altered to include a transgene comprisingDNA from a human immunoglobulin locus that can undergo functionalrearrangement. An endogenous immunoglobulin locus in a human-antibodytransgenic animal can be disrupted or deleted to eliminate the capacityof the animal to produce antibodies encoded by an endogenous gene.Suitable methods for producing human-antibody transgenic animals arewell known in the art. (See, for example, U.S. Pat. Nos. 5,939,598 and6,075,181 (Kucherlapati et al.), U.S. Pat. Nos. 5,569,825, 5,545,806,5,625,126, 5,633,425, 5,661,016, and 5,789,650 (Lonberg et al.),Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-2555 (1993),Jakobovits et al., Nature, 362: 255-258 (1993), Jakobovits et al. WO98/50433, Jakobovits et al. WO 98/24893, Lonberg et al. WO 98/24884,Lonberg et al. WO 97/13852, Lonberg et al. WO 94/25585, Lonberg et al.EP 0 814 259 A2, Lonberg et al. GB 2 272 440 A, Lonberg et al., Nature368:856-859 (1994), Lonberg et al., Int Rev Immunol 13(1):65-93 (1995),Kucherlapati et al. WO 96/34096, Kucherlapati et al. EP 0 463 151 B1,Kucherlapati et al. EP 0 710 719 A1, Surani et al. U.S. Pat. No.5,545,807, Bruggemann et al. WO 90/04036, Bruggemann et al. EP 0 438 474B1, Taylor et al., Int. Immunol. 6(4)579-591 (1994), Taylor et al.,Nucleic Acids Research 20(23):6287-6295 (1992), Green et al., NatureGenetics 7:13-21 (1994), Mendez et al., Nature Genetics 15:146-156(1997), Tuaillon et al., Proc Natl Acad Sci USA 90(8):3720-3724 (1993)and Fishwild et al., Nat Biotechnol 14(7):845-851 (1996), the teachingsof each of the foregoing are incorporated herein by reference in theirentirety.)

As described herein, human-antibody transgenic animals can be immunizedwith a suitable composition comprising an antigen of interest (e.g., arecombinant cell expressing a CXCR3 protein). Antibody producing cellscan be isolated and fused to form hybridomas using conventional methods.Hybridomas that produce human antibodies having the desiredcharacteristics (e.g., specificity, affinity) can be identified usingany suitable assay (e.g., ELISA) and, if desired, selected and subclonedusing suitable culture techniques.

Human-antibody transgenic animals provide a source of nucleic acids thatcan be enriched in nucleic acids that encode antibodies having desiredproperties, such as specificity and affinity. For example, nucleic acidsencoding antibodies or antibody variable regions can be isolated fromhuman-antibody transgenic mice that have been immunized with a CXCR3protein. The isolated nucleic acids or portions thereof (e.g., portionsencoding variable regions, CDRs, framework regions) can be expressedusing any suitable method (e.g., phage display) to produce a library ofantibodies or antigen-binding fragments of antibodies (e.g., singlechain antigen-binding fragments, double chain antigen-binding fragments)that is enriched for antibodies or antigen-binding fragments that bind aCXCR3 protein. Such a library can exhibit enhanced diversity (e.g.,combinatorial diversity through pairing of heavy chain variable regionsand light chain variable regions) relative to the repertoire ofantibodies produced in the immunized human-antibody transgenic animal.The library can be screened using any suitable assay (e.g., a CXCR3protein binding assay) to identify antibodies or antigen-bindingfragments having desired properties (e.g., specificity, affinity). Thenucleic acids encoding antibody or antigen-binding fragments havingdesired properties can be recovered using any suitable method. (See,e.g., U.S. Pat. No. 5,871,907 (Winter et al.) and U.S. Pat. No.6,057,098 (Buechler et al.), the entire teachings of each of theforegoing are incorporated herein by reference.)

An antibody of the invention can be a CDR-grafted (e.g., humanized)antibody or an antigen-binding fragment thereof. The CDRs of aCDR-grafted antibody can be derived from a suitable antibody which bindsa CXCR3 protein (referred to as a donor antibody). For example, suitableCDRs can be derived from human mAb 5H7 or human mAb 7H5 which, asdescribed herein, bind a CXCR3 protein (CD183) or from any othersuitable antibody. Other sources of suitable CDRs include natural andartificial CXCR3 protein-specific antibodies obtained from nonhumansources, such as rodent (e.g., mouse, rat, rabbit), chicken, pig, goat,non-human primate (e.g., monkey) or non-human library.

The framework regions of a CDR-grafted antibody are preferably of humanorigin, and can be derived from any human antibody variable regionhaving sequence similarity to the analogous or equivalent region (e.g.,heavy chain variable region or light chain variable region) of theantigen-binding region of the donor antibody. Other sources of frameworkregions of human origin include human variable region consensussequences. (See, e.g., Kettleborough, C. A. et al., Protein Engineering4:773-783 (1991); Carter et al., WO 94/04679; Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, U.S. Government Printing Office(1991)).

In one embodiment, the framework regions of a CDR-grafted (e.g.,humanized) antibody chain can be derived from a variable region of humanorigin having at least about 65% overall amino acid sequence identity,and preferably at least about 70% overall amino acid sequence identity,with the amino acid sequence of the variable region of the donorantibody. A suitable framework region can also be derived from anantibody of human origin having at least about 65% amino acid sequenceidentity, and preferably at least about 70%, 80%, 90% or 95% amino acidsequence identity over the length of the framework region within theamino acid sequence of the equivalent portion (e.g., framework region)of the donor antibody. For example, a suitable framework region of humanorigin can be derived from an antibody of human origin (e.g., a humanantibody) having at least about 65% amino acid sequence identity, andpreferably at least about 70%, 80%, 90% or 95% amino acid sequenceidentity, over the length of the particular framework region being used,when compared to the amino acid sequence of the equivalent portion(e.g., framework region) of the donor antibody. Amino acid sequenceidentity can be determined using a suitable amino acid sequencealignment algorithm, such as CLUSTAL W, using the default parameters.(Thompson J. D. et al., Nucleic Acids Res. 22:4673-4680 (1994).)

Framework regions of human origin can include amino acid substitutionsor replacements, such as “back mutations” which replace an amino acidresidue in the framework region of human origin with a residue from thecorresponding position of the donor antibody. One or more mutations inthe framework region can be made, including deletions, insertions andsubstitutions of one or more amino acids. Preferably, the CDR-grafted(e.g., humanized) antibody binds a CXCR3 protein with an affinitysimilar to, substantially the same as, or better than that of the donorantibody. Variants can be produced by a variety of suitable methods,including mutagenesis of nonhuman donor or acceptor human chains. (See,e.g., U.S. Pat. No. 5,693,762 (Queen et aL) and U.S. Pat. No. 5,859,205(Adair et al.), the entire teachings of which are incorporated herein byreference.)

Constant regions of antibodies, antibody chains (e.g., heavy chain,light chain) or fragments or portions thereof of the invention, ifpresent, can be derived from any suitable source. For example, constantregions of human, humanized and certain chimeric antibodies, antibodychains (e.g., heavy chain, light chain) or fragments or portionsthereof, if present can be of human origin and can be derived from anysuitable human antibody or antibody chain. For example, a constantregion of human origin or portion thereof can be derived from a human κor λ, light chain, and/or a human γ (e.g., γ1, γ2, γ3, γ4), μ, α (e.g.,α1, α2), δ or ε heavy chain, including allelic variants. In certainembodiments, the antibody or antigen-binding fragment (e.g., antibody ofhuman origin, human antibody) can include amino acid substitutions orreplacements that alter or tailor function (e.g., effector function).For example, a constant region of human origin (e.g., γ1 constantregion, γ2 constant region) can be designed to reduce complementactivation and/or Fc receptor binding. (See, for example, U.S. Pat. No.5,648,260 (Winter et aL), U.S. Pat. No. 5,624,821 (Winter et al.) andU.S. Pat. No. 5,834,597 (Tso et al.), the entire teachings of which areincorporated herein by reference.) Preferably, the amino acid sequenceof a constant region of human origin that contains such amino acidsubstitutions or replacements is at least about 95% identical over thefull length to the amino acid sequence of the unaltered constant regionof human origin, more preferably at least about 99% identical over thefull length to the amino acid sequence of the unaltered constant regionof human origin.

Humanized antibodies or antigen-binding fragments of a humanizedantibody can be prepared using any suitable method. Several such methodsare well-known in the art. (See, e.g., U.S. Pat. No. 5,225,539 (Winter),U.S. Pat. No. 5,530,101 (Queen et al.).) The portions of a humanizedantibody (e.g., CDRs, framework, constant region) can be obtained orderived directly from suitable antibodies (e.g., by de novo synthesis ofa portion), or nucleic acids encoding an antibody or chain thereofhaving the desired property (e.g., binds a CXCR3 protein) can beproduced and expressed. Humanized immunoglobulins comprising the desiredportions (e.g., CDR, FR, constant region) of human and nonhuman origincan be produced using synthetic and/or recombinant nucleic acids toprepare a nucleic acid (e.g., cDNA) encoding the desired humanizedchain. To prepare a portion of a chain, one or more stop codons can beintroduced at the desired position. For example, nucleic acid (e.g.,DNA) sequences coding for newly designed humanized variable regions canbe constructed using PCR mutagenesis methods to alter existing DNAsequences. (See, e.g., Kamman, M., et al., Nucl. Acids Res. 17:5404(1989).) PCR primers coding for the new CDRs can be hybridized to a DNAtemplate of a previously humanized variable region which is based on thesame, or a very similar, human variable region (Sato, K., et al., CancerResearch 53:851-856 (1993)). If a similar DNA sequence is not availablefor use as a template, a nucleic acid comprising a sequence encoding avariable region sequence can be constructed from syntheticoligonucleotides (see e.g., Kolbinger, F., Protein Engineering 8:971-980(1993)). A sequence encoding a signal peptide can also be incorporatedinto the nucleic acid (e.g., on synthesis, upon insertion into avector). The natural signal peptide sequence from the acceptor antibody,a signal peptide sequence from another antibody or other suitablesequence can be used (see, e.g., Kettleborough, C. A., ProteinEngineering 4:773-783 (1991)). Using these methods, methods describedherein or other suitable methods, variants can be readily produced. Inone embodiment, cloned variable regions can be mutated, and sequencesencoding variants with the desired specificity can be selected (e.g.,from a phage library; see, e.g., U.S. Pat. No. 5,514,548 (Krebber etal.) and WO 93/06213 (Hoogenboom et al.)).

The antibody of the invention can be a chimeric antibody or anantigen-binding fragment of a chimeric antibody. Preferably, thechimeric antibody or antigen-binding fragment thereof comprises avariable region of non-human origin and a constant region of humanorigin (e.g., a human constant region).

Chimeric antibodies and antigen-binding fragments of chimeric antibodiesthat bind a CXCR3 protein can be prepared using any suitable method.Several suitable methods are well-known in the art. (See, e.g., U.S.Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,116,946 (Capon etal.).) Generally, chimeric antibodies are produced by preparing, foreach of the light and heavy chain components of the chimericimmunoglobulin, a recombinant nucleic acid comprising a first nucleotidesequence encoding at least the variable region of an antibody from afirst species that binds a CXCR3 protein that is joined in-frame to asecond nucleotide sequence encoding at least a part of a constant regionfrom an antibody of a different species. Generally, the recombinantnucleic acid encodes a chimeric heavy chain or a chimeric light chain.However, if desired, a single recombinant nucleic acid encoding achimeric heavy chain and a chimeric light chain can be prepared. Therecombinant nucleic acids can be assembled in or inserted into anexpression vector. The recombinant nucleic acid(s) can be introducedinto a suitable host cell that is capable of expressing the chimericantibody or chimeric antibody chain using any suitable method (e.g.,transfection, transformation, infection) to produce a recombinant hostcell. The recombinant host cell can be maintained under conditionssuitable for expression of the chimeric antibody or chimeric antibodychain and the antibody or chain can be recovered.

Nucleic acids encoding the variable region of antibody light and heavychains can be obtained from cells (e.g., B cells, hybridoma cells) thatproduce an antibody that binds a CXCR3 protein. For example, nucleicacids that encode human heavy and light chain variable regions that canbind a CXCR3 protein can be obtained from hybridoma 5H7 and hybridoma7H5 described herein. Nucleic acids that encode constant regions can beobtained from suitable sources using any suitable technique, such asconventional techniques of recombinant DNA technology. The nucleotidesequences of nucleic acids encoding human κ or λ light chain constantregions, and γ (e.g., γ1, γ2, γ3, γ4), α (e.g., α1, α2), δ or ε humanheavy chain constant regions are readily available.

The invention also relates to a bispecific antibody or antigen-bindingfragment thereof (e.g., F(ab′)₂), which binds a CXCR3 protein and atleast one other antigen. In a particular embodiment, the bispecificantibody, or antigen-binding fragment thereof binds an epitope on aCXCR3 protein. In other embodiments, the bispecific antibody orantigen-binding fragment thereof has the epitopic specificity of humanmAb 5H7 or human mAb 7H5 and at least one other antibody. Bispecificantibodies can be secreted by triomas and hybrid hybridomas. Generally,triomas are formed by fusion of a hybridoma and a lymphocyte (e.g.,antibody secreting B cell) and hybrid hybridomas are formed by fusion oftwo hybridomas. Each of the cells that are fused to produce a trioma orhybrid hybridoma produces a monospecific antibody. However, triomas andhybrid hybridomas can produce an antibody containing antigen-bindingsites which recognize different antigens. The supernatants of triomasand hybrid hybridomas can be assayed for bispecific antibody using asuitable assay (e.g., ELISA), and bispecific antibodies can be purifiedusing conventional methods. (See, e.g., U.S. Pat. No. 5,959,084 (Ring etal.) U.S. Pat. No. 5,141,736 (Iwasa et al.), U.S. Pat. Nos. 4,444,878,5,292,668 and 5,523,210 (Paulus et al.) and U.S. Pat. No. 5,496,549(Yamazaki et al.).)

The various portions of an antibody (e.g., mouse antibody, humanantibody, humanized antibody, chimeric antibody and antigen-bindingfragments of the foregoing) can be joined together chemically usingconventional techniques, or can be prepared as a continuous polypeptidechain by expression (in vivo or in vitro) of a nucleic acid (one or morenucleic acids) encoding antibody. For example, nucleic acids encoding ahuman, humanized or chimeric chain can be expressed in vivo or in vitroto produce a continuous polypeptide chain. See, e.g., Cabilly et al.,U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European PatentNo. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S.et al., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No.5,225,539; Winter, European Patent No. 0,239,400 B1; Queen et al.,European Patent No. 0 451 216 B1; and Padlan, E. A. et al., EP 0 519 596A1. See also, Newman, R. et al., BioTechnology, 10: 1455-1460 (1992),regarding primatized antibody, and Ladner et al., U.S. Pat. No.4,946,778 and Bird, R. E. et al., Science, 242: 423-426 (1988))regarding single chain antibodies.

The invention also relates to antigen-binding fragments of antibodies,antibody chains and antigen-binding portions of antibody chains thatretain the capacity to bind antigen (e.g., a CXCR3 protein). Suchantigen-binding fragments of antibodies retain the antigen-bindingfunction of a corresponding full-length antibody (e.g., bindingspecificity for a CXCR3 protein), and preferably inhibit binding ofligand (e.g., IP-10, MIG or I-TAC) to a CXCR3 protein. Antigen-bindingfragments of antibodies encompassed by the invention include, Fvfragments (e.g., single chain Fv fragments (scFv)), Fab fragments, Fab′fragments and F(ab)₂ fragments, for example. Such antigen-bindingfragments can be produced using any suitable method, for example byenzymatic cleavage and/or using recombinant DNA technology. For example,an antibody can be cleaved with papain or pepsin to yield a Fab fragmentor F(ab′)₂ fragment, respectively. Other proteases with the requisitesubstrate specificity can also be used to generate antigen-bindingfragments of antibodies, such as Fab fragments or F(ab)₂ fragments andvariable domains (V_(H), V_(L)). Similarly, Fv fragments can be preparedby digesting an antibody with a suitable protease or using recombinantDNA technology. For example, a nucleic acid can be prepared that encodesa light chain variable region and heavy chain variable region that areconnected by a suitable peptide linker, such as a chain of two to abouttwenty glycyl residues. The nucleic acid can be introduced into asuitable host (e.g., E. coli) using any suitable technique (e.g.,transfection, transformation, infection), and the host can be maintainedunder conditions suitable for expression of a single chain Fv fragment.A variety of antigen-binding fragments of antibodies can be preparedusing antibody genes in which one or more stop codons has beenintroduced upstream of the natural stop site. For example, an expressionconstruct encoding a F(ab′)₂ portion of an immunoglobulin heavy chaincan be designed by introducing a translation stop codon at the 3′ end ofthe sequence encoding the hinge region of the heavy chain.

The invention also relates to the individual heavy and light chains ofthe antibodies (e.g., mouse antibodies, human antibodies, humanizedantibodies, chimeric antibodies) that bind a CXCR3 protein and toantigen-binding portions thereof. The heavy chains or light chains (andantigen-binding portions thereof) of the invention can bind a CXCR3protein individually, and/or when paired with a complementary light orheavy chain, respectively. Complementary chains can be identified usingany suitable method (e.g., phage display, transgenic animals). Forexample, a transgenic animal comprising a functionally rearrangednucleic acid encoding a desired heavy chain can be prepared. Theheavy-chain transgenic animal can be immunized with the antigen ofinterest and hybridomas produced. Because of allelic exclusion atimmunoglubulin loci, the heavy-chain transgenic mouse may notsignificantly express endogenous heavy chains and substantially allantibodies elicited by immunization can comprise the heavy chain ofinterest and a complementary light chain.

The antigen-binding properties (e.g., specificity, affinity) ofantibodies and antigen-binding fragments of antibodies can be elucidatedusing any suitable method. For example, binding specificity can bedetermined using assays in which formation of a complex between antibodyor antigen-binding fragment thereof and a CXCR3 protein, is detected ormeasured. Compositions which comprise a CXCR3 protein and which can beused to assess antigen-binding properties of the antibodies andantigen-binding fragments described herein include a membrane fractionof a cell comprising a CXCR3 protein, a cell bearing a CXCR3 protein,such as a human lymphocyte, human lymphocyte cell line, a recombinanthost cell comprising a nucleic acid encoding a CXCR3 protein whichexpresses a CXCR3 protein, a synthetic peptide and the like. Bindingand/or functional assays or other suitable methods also can be used inprocedures for the identification and/or isolation of antibodies (e.g.,human and/or humanized antibodies) having the requisite specificity(e.g., an assay in which binding between a cell bearing a CXCR3 proteinand a ligand thereof (e.g., IP-10, MIG or I-TAC, an immobilized IP-10,MIG or I-TAC fusion protein (e.g., IP-10-, MIG- or I-TAC-Fc fusionprotein) is detected and/or measured (e.g. in a binding assay or in anassay measuring CXCR3-mediated function, e.g. intracellular calciumrelease or chemotaxis), or another suitable method.

The antibodies of the invention bind a human CXCR3 protein. In someembodiments, the antibody binds a CXCR3 protein and inhibits binding ofa ligand, such as IP-10, MIG or I-TAC, to the CXCR3 protein. Forexample, the antibody can inhibit a CXCR3-mediated function of a cellexpressing a CXCR3 protein (e.g., signal transduction, intracellularcalcium release, chemotaxis, cell differentiation, cell proliferation orcell activation). In a preferred embodiment, the antibody of theinvention binds a CXCR3 protein and inhibits binding of at least two,preferably at least three ligands, IP-10, MIG and I-TAC, to the CXCR3protein.

Preferred antibodies which bind a CXCR3 protein include chimericantibodies, humanized antibodies and antigen-binding fragments of theforegoing. Particularly preferred antibodies are human antibodies andantigen-binding fragments of human antibodies. As described herein,human antibodies designated human mAb 5H7 and human mAb 7H5 which bind aCXCR3 protein have been produced. Both human mAb 5H7 and human mAb 7H5were produced as IgG2, kappa antibodies in the original hybridomas, asdescribed herein. The nucleic acid sequences encoding the heavy andlight chain variable regions of human mAb 5H7 were cloned into a vector,pLKTOK59(D). The resulting construct encoded an IgG1, kappa antibodywith the variable regions and binding specificity of human mAb 5H7.

=Human mAb 5H7 (IgG2, kappa) can be produced by hybridoma 5H7, alsoreferred to as hybridoma LS328-5H7-2-6, which was deposited on Aug. 7,2003, on behalf of Millennium Pharmaceuticals Inc., 75 Sidney Street,Cambridge, Mass., 02139, USA, at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110, U.S.A., under AccessionNo. PTA-5388. The invention relates to hybridoma 5H7, to the antibody itproduces, antigen-binding fragments thereof, and to nucleic acidsencoding the antibody and portions thereof (e.g., heavy chain, heavychain variable region, light chain, light chain variable region). Asdescribed herein, hybridoma 5H7 produces an IgG2, kappa antibody.

Human mAb 7H5 can be produced by hybridoma 7H5, also referred to ashybridoma LS329-7H5-2-19-7, which was deposited on Aug. 7, 2003, onbehalf of Millennium Pharmaceuticals Inc., 75 Sidney Street, Cambridge,Mass., 02139, USA, at the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110, U.S.A., under Accession No.PTA-5389. The invention relates to hybridoma 7H5, to the antibody itproduces, antigen-binding fragments thereof, and to nucleic acidsencoding the antibody and portions thereof (e.g., heavy chain, heavychain variable region, light chain, light chain variable region). Asdescribed herein, hybridoma 7H5 produces an IgG2, kappa antibody.

In one embodiment, the antibody or antigen-binding fragment thereof canbind to the same or similar epitope as human mAb 5H7 or human mAb 7H5.Antibodies and antigen-binding fragments that bind the same or similarepitope as human mAb 5H7 or human mAb 7H5 can be identified using anysuitable method, such as a competitive binding assay. For example, anantibody can be tested for the ability to competitively inhibit bindingof human mAb 5H7 or human mAb 7H5 to a CXCR3 protein expressed on thesurface of a cell. Competitive inhibition of binding of human mAb 5H7 orhuman mAb 7H5 in this type of assay is indicative that the test antibodybinds the same or similar epitope as human mAb 5H7 or human mAb 7H5.

In particular embodiments, the antibody or antigen-binding fragmentthereof can have the epitopic specificity of human mAb 5H7 or human mAb7H5. The fine epitopic specificity of an antibody can be determinedusing any suitable method, such as peptide competition or mutationalanalysis. For example, a series of CXCR3 variants comprising′amino acidreplacements can be prepared and an antibody can be tested for theability to bind each variant. Inhibited or abrogated binding to avariant comprising a particular amino acid substitution is indicativethat the substituted amino acid is part of the epitope that the antibodybinds. (See, Higgins et al., J. Biol. Chem. 275:25652-25664 (2000).)

Isolated antibodies or antigen-binding fragments of the inventiontypically bind to a CXCR3 protein with a binding equilibrium associationconstant (K_(A)) of at least about 10⁶ M⁻¹, at least about 10⁷ M⁻¹,preferably at least about 10⁸ M⁻¹, more preferably, at least about 10⁹M⁻¹, and more preferably at least about 10¹⁰ to about 10¹¹ M^(−l) orhigher.

As used herein, an “antigen-binding portion” of an antibody comprises aportion of a variable region of an antibody, said portion comprising atleast one, two, preferably three CDRs selected from CDR1, CDR2, andCDR3. The antigen-binding portion can comprise a portion of animmunoglobulin heavy chain or an immunoglobulin light chain.

In more particular embodiments, the antibody comprises one, two or threeheavy chain complementarity determining regions (HCDR1, HCDR2 and/orHCDR3) having the amino acid sequences of heavy chain CDR1, CDR2 andCDR3 of human mAb 5H7. If desired, one amino acid residue in the heavychain CDR1, one or two amino acid residues in the heavy chain CDR2,and/or one, two or three amino acid residues in CDR3 can beconservatively substituted. The antibody can further comprise one, twoor three light chain complementarity determining regions (LCDR1, LCDR2and/or LCDR3) having the amino acid sequences of light chain CDR1, CDR2and CDR3 of human mAb 5H7. If desired, one or two amino acid residues inthe light chain CDR1, one amino acid residue in the light chain CDR2and/or one or two amino acid residues in the light chain CDR3 can beconservatively substituted. Preferably, the antibody comprises the threeheavy chain CDRs and the three light chain CDRs of human mAb 5H7. Inmore particular embodiments, the antibody comprises the heavy chainvariable region of human mAb 5H7 (5H7 VH, SEQ ID NO: 2) and the lightchain variable region of human mAb 5H7 (5H7 VK, SEQ ID NO:10).

In additional particular embodiments, the antibody comprises one, two orthree heavy chain complementarity determining regions (HCDR1, HCDR2and/or HCDR3) having the amino acid sequences of the heavy chain CDR1,CDR2, and CDR3 of human mAb 7H5. If desired, one amino acid residue inthe heavy chain CDR1, one or two amino acid residues in the heavy chainCDR2 and/or one, two or three amino acid residues in the heavy chainCDR3 can be conservatively substituted. The antibody can furthercomprise one, two or three light chain complementarity determiningregions (LCDR1, LCDR2 and/or LCDR3) having the amino acid sequences ofthe light chain CDR1, CDR2, and CDR3 of human mAb 7H5. If desired, oneor two amino acid residues in the light chain CDR1, one amino acidresidue in the light chain CDR2 and/or one or two amino acid residues inthe light chain CDR3 can be conservatively substituted. Preferably, theantibody comprises the three heavy chain CDRs and the three light chainCDRs of human mAb 7H5. In more particular embodiments, the antibodycomprises the heavy chain variable region of human mAb 7H5 (7H5 VH, SEQID NO:18) and the light chain variable region of human mAb 7H5 (7H5 VK,SEQ ID NO:26).

The sequence ID numbers of the immuglobulin heavy and light chainvariable regions and respective CDRs of human mAb 5H7 and human mAb 7H5are listed below in Tables 1 and 2.

TABLE 1 Sequence ID Numbers of Variable Regions of Human Monoclonal 5H7Antibody Chain Portion Type Length SEQ ID NO: Heavy Full Length DNA 3781 (5H7 VH) Full Length aa 126 2 CDR1 DNA 15 3 CDR1 aa 5 4 CDR2 DNA 51 5CDR2 aa 17 6 CDR3 DNA 51 7 CDR3 aa 17 8 Light Full Length DNA 321 9 (5H7VK) Full Length aa 107 10 CDR1 DNA 33 11 CDR1 aa 11 12 CDR2 DNA 21 13CDR2 aa 7 14 CDR3 DNA 27 15 CDR3 aa 9 16

TABLE 2 Sequence ID Numbers of Variable Regions of Human Monoclonal 7H5Antibody Chain Portion Type Length SEQ ID NO: Heavy Full Length DNA 37817 (7H5 VH) Full Length aa 126 18 CDR1 DNA 15 19 CDR1 aa 5 20 CDR2 DNA51 21 CDR2 aa 17 22 CDR3 DNA 51 23 CDR3 aa 17 24 Light Full Length DNA321 25 (7H5 VK) Full Length aa 107 26 CDR1 DNA 33 27 CDR1 aa 11 28 CDR2DNA 21 29 CDR2 aa 7 30 CDR3 DNA 27 31 CDR3 aa 9 32

In additional embodiments, the invention provides novel heavy chains andlight chains of the antibodies and antigen-binding fragments describedherein. In particular embodiments, the antibody heavy chains orantigen-binding portions thereof comprise at least one, two, preferablythree CDRs having amino acid sequences of the heavy chain CDRs of humanmAb 5H7 or the heavy chain CDRs of human mAb 7H5. For example, theantibody heavy chains or antigen-binding portions thereof can comprisean amino acid sequence selected from the group consisting of SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:22, and SEQ IDNO:24. If desired, residues in each heavy chain CDR can beconservatively substituted as described above. In preferred embodiments,the antibody heavy chains or antigen-binding portions thereof comprisethree CDRs which have the amino acid sequences of the three CDRs of theheavy chain of human mAb 5H7 or the three CDRs of the heavy chain ofhuman mAb 7H5. In other embodiments, the antibody heavy chains orantigen-binding portions thereof comprise the heavy chain variableregion of human mAb 5H7 or human mAb 7H5. For example, the antibodyheavy chains can comprise an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2 and SEQ ID NO: 18. The antibody heavy chainsand portions thereof can comprise any suitable framework regions and/orconstant region.

In certain embodiments, the antibody light chains or antigen-bindingportions thereof comprise at least one, two, preferably three CDRshaving amino acid sequences of the light chain CDRs of human mAb 5H7 orthe light chain CDRs of human mAb 7H5. For example, the antibody lightchains or antigen-binding portions thereof can comprise an amino acidsequence selected from the group consisting of SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:32. Ifdesired, residues in each light chain CDR can be conservativelysubstituted as described above. In preferred embodiments, the antibodylight chains or antigen-binding portions thereof comprise three CDRswhich have the amino acid sequences of the three CDRs of the light chainof human mAb 5H7 or the three CDRs of the light chain of human mAb 7H5.In other embodiments, the antibody light chains or antigen-bindingportions thereof comprise the light chain variable region of human mAb5H7 or human mAb 7H5. For example, the antibody light chains cancomprise an amino acid sequence selected from the group consisting ofSEQ ID NO: 10 and SEQ ID NO: 26. The antibody light chains and portionsthereof can comprise any suitable framework regions and/or constantregion.

Fusion Proteins and Immuno-Conjugates

Fusion proteins and immuno-conjugates can be produced in which anantibody moiety (e.g., antibody or antigen-binding fragment thereof,antibody chain or antigen-binding portion thereof) is linked directly orindirectly to a non-immunoglobulin moiety (i.e., a moiety which does notoccur in immunoglobulins as found in nature). Fusion proteins comprisean antibody moiety and a non-immunoglobulin moiety that are componentsof a single continuous polypeptide chain. The non-immunoglobulin moietycan be located N-terminally, C-terminally or internally with respect tothe antibody moiety. For example, some embodiments can be produced bythe insertion of a nucleic acid encoding immunoglobulin sequences into asuitable expression vector, such as a pET vector (e.g., pET-15b,Novagen), a phage vector (e.g., pCANTAB 5 E, Pharmacia), or other vector(e.g., pRIT2T Protein A fusion vector, Pharmacia). The resultingconstruct can be expressed (e.g., in vivo by a suitable host cell, invitro) to produce antibody chains that comprise a non-immunoglobulinmoiety (e.g., Histidine tag, E tag, Protein A IgG binding domain).Fusion proteins can be isolated or recovered using any suitabletechnique, such as chromatography using a suitable affinity matrix (seee.g., Current Protocols in Molecular Biology (Ausubel, F. M. et al.,eds., Vol. 2, Suppl. 26, pp. 16.4.1-16.7.8 (1991)).

In other embodiments, the antibody moiety and non-immunoglobulin moietymay not be part of a continuous polypeptide chain, but can be connectedor conjugated directly or indirectly through any suitable linker.Suitable methods for connecting or conjugating the moieties are wellknown in the art. (See, e.g., Ghetie et al., Pharmacol. Ther. 63:209-34(1994)). A variety of suitable linkers (e.g., heterobifunctionalreagents) and methods for preparing immuno-conjugates are well known inthe art. (See, for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996).) The non-immunoglobulin moietycan be bonded to a chemically reactive group on the antibody moiety,e.g., to a free amino, imino, hydroxyl, thiol or carboxyl group (e.g.,the N- or C-terminus, to the epsilon amino group of one or more lysineresidue or to the sulfhydryl of one or more cysteinyl residue). The siteto which the non-immunoglobulin moiety is bound can be a natural residuein the amino acid sequence of the antibody moiety or it can beintroduced in the antibody moiety, e.g., by DNA recombinant technology(e.g., by introducing a cysteine or a protease cleavage site in theamino acid sequence) or by protein biochemistry, e.g., reduction, pHadjustment or proteolysis. The immuno-conjugate can be purified fromreactants by employing methodologies, e.g., column chromatography (e.g.,affinity chromatography, ion exchange chromatography, gel filtration,hydrophobic interaction chromatography), dialysis, diafiltration orprecipitation, well known to those of skill in the art. Theimmuno-conjugate can be evaluated by employing methodologies, e.g.,SDS-PAGE, mass spectroscopy or capillary electrophoresis, well known tothose skilled in the art.

Suitable non-immunoglobulin moieties for inclusion in animmuno-conjugate include a therapeutic moiety, such as a toxin (e.g.,cytotoxin, cytotoxic agent), a therapeutic agent (e.g., achemotherapeutic agent), an antimetabolite, an alkylating agent, ananthracycline, an antibiotic, an anti-mitotic agent, a biologicalresponse modifier (e.g., a cytokine (e.g., an interleukin, aninterferon, a tumor necrosis factor), a growth factor (e.g., aneurotrophic factor)), a plasminogen activator, a radionuclide (e.g., aradioactive ion), an enzyme) or a detectable label, e.g., a radionuclide(e.g., technetium-99m, indium-111), and the like. Another suitablenon-immunoglobulin moiety for inclusion in an immuno-conjugate includesan organic moiety intended to increase the in vivo serum half-life ofthe antibody.

Suitable cytotoxins or cytotoxic agents include any agent that isdetrimental to cells. Examples of suitable cytotoxins or cytotoxicagents include cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin (e.g., mitomycin C), etoposide, tenoposide, doxorubicin,daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, puromycin, and analogs or homologsof any of the foregoing agents.

Suitable therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepachlorambucil, CC-1065, melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, colchicines, TAXOL® (paclitaxel,Bristol-Myers Squibb Company) and maytansinoids (e.g., maytansinol (seeU.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092,5,585,499, 5,846,545), DM1)). Suitable radionuclide therapeutic agentsinclude, for example iodine (e.g., iodine-125, -126), yttrium (e.g.,yttrium-90, -91) and praseodymium (e.g., praseodymium-144, -145).

In certain embodiments, the therapeutic agent can be a protein orpolypeptide possessing a desired biological activity. Such proteins orpolypeptides can include, for example, a toxin such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as a tumornecrosis factor (e.g., TNFα, TNFβ), and interferon (e.g., α-interferon,β-interferon, γ-interferon), a neurotrophic factor (e.g., nerve growthfactor), a growth factor (e.g., platelet derived growth factor), aplasminogen activator (e.g., tissue plasminogen activator); orbiological response modifiers such as, for example, cytokines andlymphokines, (e.g., interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”)), or othergrowth factors. In other embodiments, the antibody or antigen-bindingfragment of the invention can be conjugated to a second antibody orantigen-binding fragment to form an antibody heteroconjugate. (See,e.g., U.S. Pat. No. 4,676,980 (Segal).)

Suitable organic moieties intended to increase the in vivo serumhalf-life of the antibody can include one, two or more linear orbranched moiety selected from a hydrophilic polymeric group (e.g., alinear or a branched polymer (e.g., a polyalkane glycol such aspolyethylene glycol, monomethoxy-polyethylene glycol and the like), acarbohydrate (e.g., a dextran, a cellulose, a polysaccharide and thelike), a polymer of a hydrophilic amino acid (e.g., polylysine,polyaspartate and the like), a polyalkane oxide and polyvinylpyrolidone), a fatty acid group (e.g., a mono-carboxylic acid or adi-carboxylic acid), a fatty acid ester group, a lipid group (e.g.,diacylglycerol group, sphingolipid group (e.g., ceramidyl)) or aphosopholipid group (e.g., phosphatidyl ethanolamine group). Preferably,the organic moiety is bound to a predetermined site where the organicmoiety does not impair the function (e.g., decrease the antigen bindingaffinity) of the resulting immuno-conjugate compared to thenon-conjugated antibody moiety. The organic moiety can have a molecularweight of about 500 Da to about 50,000 Da, preferably about 2000, 5000,10,000 or 20,000 Da. Examples and methods for modifying polypeptides,e.g., antibodies, with organic moieties can be found, for example, inU.S. Pat. Nos. 4,179,337 and 5,612,460, PCT Publication Nos. WO 95/06058and WO 00/26256, and U.S. Patent Application Publication No.20030026805, the entire teachings of which are incorporated herein byreference.

Nucleic Acids and Constructs

The present invention also relates to isolated and/or recombinant(including, e.g., essentially pure) nucleic acids comprising sequenceswhich encode an antibody or antigen-binding fragment (e.g., a human,humanized, chimeric antibody or light or heavy chain of any of theforegoing) or fusion protein of the invention.

Nucleic acids referred to herein as “isolated” are nucleic acids whichhave been separated away from other material (e.g., other nucleic acidssuch as genomic DNA, cDNA and/or RNA) in its original environment (e.g.,in cells or in a mixture of nucleic acids such as a library). Anisolated nucleic acid can be isolated as part of a vector (e.g., aplasmid). Nucleic acids can be naturally occurring, produced by chemicalsynthesis, by combinations of biological and chemical methods (e.g.,semisynthetic), and be isolated using any suitable methods.

Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, includingmethods which rely upon artificial recombination, such as cloning into avector or chromosome using, for example, restriction enzymes, homologousrecombination, viruses and the like, and nucleic acids prepared usingthe polymerase chain reaction (PCR). “Recombinant” nucleic acids arealso those that result from recombination of endogenous or exogenousnucleic acids through the natural mechanisms of cells or cells modifiedto allow recombination (e.g., cells modified to express Cre or othersuitable recombinase), but are selected for after the introduction tothe cells of nucleic acids designed to allow and make recombinationprobable. For example, a functionally rearranged human-antibodytransgene is a recombinant nucleic acid.

The present invention also relates more specifically to nucleic acidsthat encode the heavy chains and/or light chains of the antibodies andantigen-binding portions described herein. For example, in oneembodiment, the nucleic acid can encode a heavy chain or antigen-bindingportion thereof that comprises at least one, two or preferably threeCDRs having the amino acid sequences of the heavy chain CDRs of humanmAb 5H7 wherein, optionally, one, two or three amino acids in each CDRcan be conservatively substituted as described above. In anotherembodiment, the nucleic acid can encode a heavy chain or antigen-bindingportion thereof that comprises at least one, two or preferably threeCDRs having the amino acid sequences of the heavy chain CDRs of humanmAb 7H5 wherein, optionally, one, two or three amino acids in each CDRcan be conservatively substituted as described above. In preferredembodiments, the nucleic acid encodes an antibody heavy chain orantigen-binding portion thereof that comprises three CDRs that have theamino acid sequences of the three CDRs of the heavy chain of human mAb5H7 or the three CDRs of the heavy chain of human mAb 7H5. In otherembodiments, the nucleic acid encodes an antibody heavy chain orantigen-binding portion thereof that comprises the heavy chain variableregion of human mAb 5H7 or human mAb 7H5. For example, the nucleic acidcan comprise a nucleotide sequence selected from the group consisting ofSEQ ID NO: 1 and SEQ ID NO: 17. The antibody heavy chains and portionsthereof can further comprise any suitable framework regions and/orconstant region.

In another embodiment, the nucleic acid can encode a light chain orantigen-binding portion thereof that comprises at least one, two,preferably three CDRs having the amino acid sequences of the light chainCDRs of human mAb 5H7 wherein, optionally, one or two amino acids ineach CDR can be conservatively substituted as described above. Inanother embodiment, the nucleic acid can encode a light chain orantigen-binding portion thereof that comprises at least one, two,preferably three CDRs having the amino acid sequences of the light chainCDRs of human mAb 7H5 wherein, optionally, one or two amino acids ineach CDR can be conservatively substituted as described above. Inpreferred embodiments, the nucleic acid encodes an antibody light chainor antigen-binding portion thereof which comprises three CDRs which havethe amino acid sequences of the three CDRs of the light chain of humanmAb 5H7 or the three CDRs of the light chain of human mAb 7H5. In otherembodiments, the nucleic acid encodes an antibody light chain orantigen-binding portion thereof which comprises the light chain variableregion of human mAb 5H7 or human mAb 7H5. For example, the nucleic acidcan comprise a nucleotide sequence selected from the group consisting ofSEQ ID NO: 9 and SEQ ID NO: 25. The antibody light chains and portionsthereof can comprise any suitable framework regions and/or constantregion.

Nucleic acid molecules of the present invention can be used in theproduction of antibodies (e.g., human antibodies, humanized antibodies,chimeric antibodies and antigen-binding fragments of the foregoing) thatbind a CXCR3 protein. For example, a nucleic acid (e.g., DNA) encodingan antibody of the invention can be incorporated into a suitableconstruct (e.g., an expression vector (e.g., a pLKTOK59 vector)) forfurther manipulation or for production of the encoded polypeptide insuitable host cells.

Expression constructs or expression vectors suitable for the expressionof an antibody or antigen-binding fragment that binds a CXCR3 proteinare also provided. For example, a nucleic acid encoding all or part of adesired antibody can be inserted into a nucleic acid vector, such as aplasmid or virus, for expression. The vector can be capable ofreplication in a suitable biological system (e.g., a replicon). Avariety of suitable vectors are known in the art, including vectorswhich are maintained in single copy or multiple copies, or which becomeintegrated into the host cell chromosome.

Suitable expression vectors can contain a number of components, forexample, an origin of replication, a selectable marker gene, one or moreexpression control elements, such as a transcription control element(e.g., promoter, enhancer, terminator) and/or one or more translationsignals, a signal sequence or leader sequence, and the like. Expressioncontrol elements and a signal or leader sequence, if present, can beprovided by the vector or other source. For example, the transcriptionaland/or translational control sequences of a cloned nucleic acid encodingan antibody chain can be used to direct expression.

A promoter can be provided for expression in a desired host cell.Promoters can be constitutive or inducible. For example, a promoter canbe operably linked to a nucleic acid encoding an antibody, antibodychain or portion thereof, such that it directs transcription of thenucleic acid. A variety of suitable promoters for prokaryotic (e.g.,lac, tac, T3, T7 promoters for E. coli) and eukaryotic (e.g., simianvirus 40 early or late promoter, Rous sarcoma virus long terminal repeatpromoter, cytomegalovirus promoter, adenovirus late promoter, EF-1apromoter) hosts are available.

In addition, expression vectors typically comprise a selectable markerfor selection of host cells carrying the vector, and, in the case of areplicable expression vector, an origin or replication. Genes encodingproducts which confer antibiotic or drug resistance are commonselectable markers and may be used in prokaryotic (e.g., β-lactamasegene (ampicillin resistance), Tet gene for tetracycline resistance) andeukaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolicacid), ampicillin, or hygromycin resistance genes). Dihydrofolatereductase marker genes permit selection with methotrexate in a varietyof hosts. Genes encoding the gene product of auxotrophic markers of thehost (e.g., LEU2, URA3, HIS3) are often used as selectable markers inyeast. Use of viral (e.g., baculovirus) or phage vectors, and vectorswhich are capable of integrating into the genome of the host cell, suchas retroviral vectors, are also contemplated.

Suitable expression vectors for expression in mammalian cells include,for example, pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, pEF-1 (InvitrogenLife Technologies, Carlsbad, Calif.), pCMV-SCRIPT®, pFB, pSG5, pXT1(Stratagene, La Jolla, Calif.), pCDEF3 (Goldman, L. A., et al.,Biotechniques, 21:1013-1015 (1996)), pSVSPORT (GIBCO division ofInvitrogen Life Technologies, Carlsbad, Calif.), pEF-Bos (Mizushima, S.,et al., Nucleic Acids Res., 18:5322 (1990)) and the like. Expressionvectors which are suitable for use in various expression hosts, such asprokaryotic cells (E. coli), insect cells (Drosophila Schnieder S2cells, Sf9) and yeast (P. methanolica, P. pastoris, S. cerevisiae) arealso available.

Thus, the invention provides an expression vector comprising a nucleicacid encoding an antibody, antigen-binding fragment of an antibody(e.g., a human, humanized, chimeric antibody or antigen-binding fragmentof any of the foregoing), antibody chain (e.g., heavy chain, lightchain) or antigen-binding portion of an antibody chain that binds aCXCR3 protein.

Recombinant Host Cells and Methods of Production

In another aspect, the invention relates to recombinant host cells and amethod of preparing an antibody or antigen-binding fragment, antibodychain (e.g., heavy chain, light chain) or antigen-binding portion of anantibody chain, or fusion protein of the invention. As used herein,“recombinant host cell” and “host cell” do not include a recombinantcell that is part of a transgenic human or isolated from a transgenichuman. The antibody or antigen-binding fragment can be obtained, forexample, by the expression of one or more recombinant nucleic acids,encoding an antibody, antigen-binding fragment of an antibody, antibodychain or antigen-binding portion of an antibody chain that binds a CXCR3protein, in a suitable host cell, or using other suitable methods. Forexample, the expression constructs described herein can be introducedinto a suitable host cell, and the resulting cell can be maintained(e.g., in culture, in an animal, in a plant) under conditions suitablefor expression of the constructs. Suitable host cells can beprokaryotic, including bacterial cells such as E. coli, B. subtilisand/or other suitable bacteria; eukaryotic cells, such as fungal oryeast cells (e.g., Pichia pastoris, Aspergillus sp., Saccharomycescerevisiae, Schizosaccharomyces pombe, Neurospora crassa), or otherlower eukaryotic cells, and cells of higher eukaryotes such as thosefrom insects (e.g., Drosophila Schnieder S2 cells, Sf9 insect cells (WO94/26087 (O'Connor)), mammals (e.g., COS cells, such as COS-1 (ATCCAccession No. CRL-1650) and COS-7 (ATCC Accession No. CRL-1651), CHO(e.g., ATCC Accession No. CRL-9096), 293 (ATCC Accession No. CRL-1573),HeLa (ATCC Accession No. CCL-2), CV1 (ATCC Accession No. CCL-70), WOP(Dailey, L., et al., J. Virol., 54:739-749 (1985), 3T3, 293T (Pear, W.S., et al., Proc. Natl. Acad. Sci. U.S.A., 90:8392-8396 (1993)) NSOcells, SP2/0, HuT 78 cells and the like, or plants (e.g., tobacco).(See, for example, Ausubel, F. M. et al., eds. Current Protocols inMolecular Biology, Greene Publishing Associates and John Wiley & SonsInc. (1993).)

The invention also relates to a recombinant host cell which comprises a(one or more) recombinant nucleic acid or expression constructcomprising a nucleic acid encoding an antibody, antigen-binding fragmentof an antibody (e.g., a human, humanized, chimeric antibody orantigen-binding fragment of any of the foregoing), antibody chain (e.g.,heavy chain, light chain), antigen-binding portion of an antibody chainthat binds a CXCR3 protein or fusion protein. In particular embodiments,the recombinant host cell is hybridoma 5H7, hybridoma 7H5.

The invention also includes a method of preparing an antibody,antigen-binding fragment of an antibody (e.g., a human, humanized,chimeric antibody or antigen-binding fragment of any of the foregoing),antibody chain (e.g., heavy chain, light chain), antigen-binding portionof an antibody chain that binds a CXCR3 protein, or fusion protein,comprising maintaining a recombinant host cell of the invention underconditions appropriate for expression of an antibody, antigen-bindingfragment of an antibody, antibody chain or antigen-binding fragment ofan antibody chain. The method can further comprise the step of isolatingor recovering the antibody, antigen-binding fragment of an antibody,antibody chain or antigen-binding fragment of an antibody chain, ifdesired.

For example, a nucleic acid molecule (i.e., one or more nucleic acidmolecules) encoding the heavy and light chains of a human antibody thatbinds a CXCR3 protein, or an expression construct (i.e., one or moreconstructs) comprising such nucleic acid molecule(s), can be introducedinto a suitable host cell to create a recombinant host cell using anymethod appropriate to the host cell selected (e.g., transformation,transfection, electroporation, infection), such that the nucleic acidmolecule(s) are operably linked to one or more expression controlelements (e.g., in a vector, in a construct created by processes in thecell, integrated into the host cell genome). The resulting recombinanthost cell can be maintained under conditions suitable for expression(e.g., in the presence of an inducer, in a suitable non-human animal, insuitable culture media supplemented with appropriate salts, growthfactors, antibiotics, nutritional supplements, etc.), whereby theencoded polypeptide(s) are produced. If desired, the encoded protein canbe isolated or recovered (e.g., from the animal, the host cell, medium,milk). This process encompasses expression in a host cell of atransgenic non-human animal (see, e.g., WO 92/03918, GenPharmInternational) or plant.

The antibodies, antigen-binding fragments, antibody chains andantigen-binding portions thereof described herein also can be producedin a suitable in vitro expression system, by chemical synthesis or byany other suitable method.

Diagnostic and Therapeutic Methods

The antibodies (including antigen-binding fragments), immunoglobulinchains (including antigen-binding portions), fusion proteins andimmuno-conjugates described herein can bind a CXCR3 protein and can beused to detect, measure, select, isolate and/or purify a CXCR3 proteinor variants thereof (e.g., by affinity purification or other suitablemethods), and to study CXCR3 protein structure (e.g., conformation) andfunction. The antibodies, immunoglobulin chains, fusion proteins andimmuno-conjugates of the present invention also can be used indiagnostic applications (e.g., in vitro, ex vivo) and/or in therapeuticapplications.

The antibodies (including antigen-binding fragments), immunoglobulinchains (including antigen-binding portions), fusion proteins andimmuno-conjugates can be used to detect and/or measure the level of aCXCR3 protein in a sample (e.g., tissue or body fluid, such as aninflammatory exudate, bronchial lavage, blood, serum, bowel fluid,biopsy). In one example, a sample (e.g., tissue and/or body fluid) canbe obtained from an individual and a suitable immunological method canbe used to detect and/or measure CXCR3 protein expression. Suitableimmunological methods for detecting or measuring CXCR3 proteinexpression include enzyme-linked immunosorbent assays (ELISA),radioimmunoassay, immunohistology, flow cytometry, and the like.

In one embodiment, the invention is a method of detecting or measuring aCXCR3 protein in a sample (e.g., a biological sample) comprisingcontacting a sample (e.g., a biological sample) with an antibody orantigen-binding fragment thereof that binds a CXCR3 protein underconditions suitable for binding of the antibody or antigen-bindingfragment to the CXCR3 protein and detecting and/or measuring binding ofthe antibody or antigen-binding fragment to the CXCR3 protein. Bindingof the antibody or antigen-binding fragment thereof to the CXCR3 proteinindicates the presence of the CXCR3 protein in the sample. In anapplication of the method, an antibody or antigen-binding fragment ofthe invention can be used to analyze normal versus inflamed tissues(e.g., from a human) for CXCR3 protein reactivity and/or expression todetect associations between disease (e.g., inflammatory bowel disease,graft rejection) and increased expression of a CXCR3 protein (e.g., inaffected tissues). In embodiments where the antibody or antigen-bindingfragment binds a CXCR3 protein, the antibodies, antigen-bindingfragments, fusion proteins and immuno-conjugates of the invention can beused to detect, measure, select, isolate and/or purify a CXCR3 proteinor a cell expressing a CXCR3 protein.

The antibodies (including antigen-binding fragments), immunoglobulinchains (including antigen-binding portions), fusion proteins andimmuno-conjugates of the present invention permit assessment of thepresence of a CXCR3 protein in normal versus inflamed tissues, throughwhich the presence or severity of disease, disease progress and/or theefficacy of therapy can be assessed. For example, therapy can bemonitored and efficacy assessed. In one example, a CXCR3 protein can bedetected and/or measured in a first sample obtained from a subjecthaving an inflammatory disease and therapy can be initiated. Later, asecond sample can be obtained from the subject and CXCR3 protein in thesample can be detected and/or measured. A decrease in the quantity ofCXCR3 protein detected or measured in the second sample can beindicative of therapeutic efficacy.

The antibodies (including antigen-binding fragments), immunoglobulinchains (including antigen-binding portions), fusion proteins andimmuno-conjugates described herein can modulate an activity or functionof a CXCR3 protein, such as ligand binding (e.g., binding of IP-10, MIGor I-TAC) and/or leukocyte infiltration function, including recruitmentand/or accumulation of leukocytes (e.g., T cells, NK cells, eosinophils)in tissues. Antibodies, immunoglobulin chains, fusion proteins andimmuno-conjugates that bind an epitope can be used to selectively targetcells expressing a CXCR3 protein for therapy. For example, an antibodythat binds an epitope on a CXCR3 protein and is capable of activatingcomplement (e.g., a human IgG1 antibody) can be administered toselectively deplete cells expressing a CXCR3 protein through, forexample, complement-mediated lysis.

Preferably the antibodies (including antigen-binding fragments),immunoglobulin chains (including antigen-binding portions), fusionproteins and immuno-conjugates can selectively bind a CXCR3 protein andinhibit one or more CXCR3-mediated functions, such as CXCR3-mediatedsignal transduction, intracellular calcium release (calcium flux),chemotaxis, cell differentiation, cell proliferation or cell activation.In particularly preferred embodiments, the antibodies, immunoglobulinchains, fusion proteins and immuno-conjugates can inhibit theinteraction of a CXCR3 protein with one or more of IP-10, MIG and I-TAC.

The antibodies (including antigen-binding fragments), immunoglobulinchains (including antigen-binding portions), fusion proteins andimmuno-conjugates described herein can be administered to a subject tomodulate an inflammatory response or to treat an inflammatory disease ordisorder. For example, an antibody which inhibits the binding of a CXCR3protein to a ligand (i.e., one or more ligands) can be administered inthe treatment of diseases associated with leukocyte (e.g., lymphocyte, Thelper-1 (Th1) lymphocyte, NK cell, eosinophil) infiltration of tissues,e.g., of mucosal tissues. An effective amount of an antibody, fusionprotein and/or immuno-conjugate (i.e., one or more) can be administeredto a subject (e.g., a mammal, such as a human or other primate) in orderto treat such a disease. For example, inflammatory diseases, including(e.g., a disease or condition mediated by a cell expressing CXCR3 (e.g.,a T helper-1 lymphocyte, an eosinophil), a mucosal inflammatory disease(e.g., inflammatory bowel disease (e.g., ulcerative colitis, Crohn'sdisease)), an autoimmune disease (e.g. rheumatoid arthritis, juvenilerheumatoid arthritis, multiple sclerosis, Graves' disease, diabetes),cancer (e.g. lymphoproliferative diseases), or an inflammatory diseaseprecipitated by foreign matter (e.g. graft rejection (e.g., allograftrejection, xenograft rejection, graft-versus-host disease), response tobacterial or viral infection, a respiratory inflammatory disease (e.g.chronic obstructive pulmonary disease)) can be treated according to thepresent invention.

According to the method, the severity of symptoms associated with aninflammatory condition can be inhibited (reduced) in whole or in part.When the subject has a relapsing or chronic condition, an effectiveamount of an antibody, fusion protein and/or immuno-conjugate of theinvention can be administered to treat the subject, and therapy can becontinued (maintenance therapy) with the same or different dosing asindicated, to inhibit relapse or renewed onset of symptoms. Preferably,the antibodies, fusion proteins and/or immuno-conjugates areadministered to treat a subject having an inflammatory disease, such asan inflammatory disease of the respiratory tract (e.g. bronchus, lung),of the nervous system (e.g., in microglia, astrocytes, in thecerebrospinal fluid), of the musculoskeletal system (e.g., in the joint)or of the alimentary canal and associated organs and tissues (e.g.mouth, salivary glands, small intestine, colon, pancreas, liver), totreat a subject rejecting an allograft or to treat a subject having acancer (e.g., of lymphocytic origin).

In a preferred embodiment, the subject to be treated has an inflammatorybowel disease (IBD), such as ulcerative colitis, Crohn's disease,ileitis, Celiac disease, nontropical Sprue, enteropathy associated withseronegative arthropathies, colitis (e.g., microscopic or collagenouscolitis), gastroenteritis (e.g., eosinophilic gastroenteritis), orpouchitis resulting after proctocolectomy and ileoanal anastomosis. Inanother preferred embodiment, the subject to be treated has anautoimmune disease affecting the nervous system (e.g., multiplesclerosis). In another preferred embodiment, the subject to be treatedhas a musculoskeletal inflammatory disease, such as rheumatoidarthritis.

In another embodiment, the subject to be treated has a pulmonaryinflammatory disease, such as a chronic obstructive lung disease (e.g.,chronic bronchitis, asthma, silicosis, chronic obstructive pulmonarydisease), hypersensitivity pneumonitis, pulmonary fibrosis (e.g.,idiopathic pulmonary fibrosis) or sarcoidosis. Subjects havingpancreatitis or insulin-dependent diabetes mellitus, Sjogren's syndromeor Behcet's syndrome can also be treated using the present method. Inother embodiments, the subject to be treated has an oral inflammatorydisease (e.g., periodontitis) or a skin inflammatory disease (e.g.,psoriasis). In other embodiments, the subject to be treated has achronic inflammatory disease, such as resulting from a bacterial orviral infection (e.g., tuberculosis or hepatitis C). In otherembodiments, the subject to be treated has a cancer, such as alymphoproliferative disease (e.g., B cell chronic lymphocytic leukemia,mucosa-associated lymphoid tissue type lymphoma, multiple myeloma,lymphoproliferative disease of granular lymphocytes).

In another embodiment, the invention is a method of inhibiting graftrejection (e.g., allograft rejection, xenograft rejection) or graftversus host disease, comprising administering to a subject in needthereof an effective amount of an antibody, fusion protein and/orimmuno-conjugate of the invention. In particular embodiments, thetransplanted graft is a cardiac or coronary tissue, a lung, a kidney,bone marrow tissue and the like.

As used herein, “subject” refers to humans and animals such as mammals,including, primates, cows, sheep, goats, horses, dogs, cats, rabbits,guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline,rodent or murine species.

Diseases and conditions associated with inflammation, infection, andcancer can be treated using the method. In a preferred embodiment, thedisease or condition is one in which the actions of cells bearing aCXCR3 protein, such as lymphocytes (e.g., activated or stimulated Tlymphocytes, NK cells) and/or eosinophils, are to be inhibited orpromoted for therapeutic or prophylactic purposes.

Diseases or conditions, including chronic diseases, of humans or otherspecies which can be treated with the antibodies, fusion proteins and/orimmuno-conjugates of the invention, include, but are not limited to:

-   -   inflammatory or allergic diseases and conditions, including        systemic anaphylaxis or hypersensitivity responses, drug        allergies (e.g., to penicillin, cephalosporins), insect sting        allergies; inflammatory bowel diseases, such as Crohn's disease,        ulcerative colitis, celiac disease, ileitis and enteritis;        sarcoidosis; vaginitis; psoriasis and inflammatory dermatoses        such as dermatitis, eczema, atopic dermatitis, allergic contact        dermatitis, urticaria; vasculitis (e.g., necrotizing, cutaneous,        and hypersensitivity vasculitis); spondyloarthropathies;        scleroderma; respiratory allergic diseases such as asthma,        allergic rhinitis, hypersensitivity lung diseases,        hypersensitivity pneumonitis, interstitial lung diseases (ILD)        (e.g., idiopathic pulmonary fibrosis, or ILD associated with        rheumatoid arthritis, or other autoimmune conditions);    -   autoimmune diseases, such as arthritis (e.g., rheumatoid        arthritis, psoriatic arthritis), multiple sclerosis, systemic        lupus erythematosus, myasthenia gravis, diabetes, including        diabetes mellitus and juvenile onset diabetes,        glomerulonephritis and other nephritides, autoimmune        thyroiditis, Behcet's syndrome, Sydenham's chorea, autoimmune        autonomic neuropathy;    -   graft rejection (e.g., in transplantation), including allograft        rejection, xenograft rejection or graft-versus-host disease;    -   viral infection, e.g., infection by hepatitis C virus (HCV),        human papilloma virus (HPV), respiratory syncytial virus,        influenza virus, simian immunodeficiency virus (SIV) or human        immunodeficiency virus (HIV);    -   cancers and/or neoplastic diseases, such as leukemias and        lymphomas;    -   other diseases or conditions in which undesirable inflammatory        responses are to be inhibited can be treated, including, but not        limited to, atherosclerosis (e.g., transplant accelerated        atherosclerosis), restenosis, cytokine-induced toxicity,        myositis (including polymyositis, dermatomyositis).

Modes of Administration

According to the method, an (i.e., one or more) antibody,antigen-binding fragment thereof, immunoglobulin heavy chain,antigen-binding portion thereof, immunoglobulin light chain,antigen-binding portion thereof, fusion protein and/or immuno-conjugatecan be administered to the subject by an appropriate route, either aloneor in combination with another drug. An “effective amount” of antibody,antigen-binding fragment thereof, immunoglobulin heavy chain,antigen-binding portion thereof, immunoglobulin light chain,antigen-binding portion thereof, fusion protein and/or immuno-conjugateis administered. An “effective amount” is an amount sufficient toachieve the desired therapeutic or prophylactic effect, under theconditions of administration, such as an amount sufficient to inhibitbinding of CXCR3 protein to a ligand, e.g., IP-10, MIG or I-TAC, andthereby, inhibit CXCR3-mediated function, such as signal transduction,intracellular calcium release, chemotaxis, cell differentiation, cellproliferation or cell activation. The antibody, antigen-binding fragmentthereof, immunoglobulin heavy chain, antigen-binding portion thereof,immunoglobulin light chain, antigen-binding portion thereof, fusionprotein and/or immuno-conjugate can be administered in a single dose ormultiple doses. Administration of the antibody, antigen-binding fragmentthereof, immunoglobulin heavy chain, antigen-binding portion thereof,immunoglobulin light chain, antigen-binding portion thereof, fusionprotein and/or immuno-conjugate can occur daily, weekly, biweekly ormonthly, preferably weekly, biweekly or monthly. Administration of theantibody, antigen-binding fragment thereof, immunoglobulin heavy chain,antigen-binding portion thereof, immunoglobulin light chain,antigen-binding portion thereof, fusion protein and/or immuno-conjugatealso can occur every other month or every three months. The antibody,antigen-binding fragment thereof, immunoglobulin heavy chain,antigen-binding portion thereof, immunoglobulin light chain,antigen-binding portion thereof, fusion protein and/or immuno-conjugatecan be administered as a bolus and/or infusion (e.g., continuousinfusion). The dosage can be determined by methods known in the art andis dependent, for example, upon the antibody, antigen-binding fragment,fusion protein and/or immuno-conjugate chosen, the subject's age,sensitivity and tolerance to drugs, and overall well-being. Typically,an effective amount can range from about 1 or 10 mg per administrationto about 800, 1000, 1500 or 2000 mg per administration for an adult.Preferably, the dosage ranges from about 10, 20, 30 or 50 mg peradministration to about 700 or 1000 mg per administration. For example,an effective amount of a human, humanized or chimeric antibody peradministration (or antigen-binding fragment of any of the foregoing) canrange from about 0.01 mg/kg to about 5, 10, 15 or 20 mg/kg, preferablyin the range of about 3, 5, 7, 10 or 15 mg/kg. Human, humanized andchimeric antibodies can often be administered with less frequency thanother types of therapeutics.

A variety of routes of administration are possible including, forexample, oral, dietary, topical, transdermal, rectal, parenteral (e.g.,intravenous, intraarterial, intramuscular, subcutaneous, intradermal,intraperatoneal injection), and inhalation (e.g., intrabronchial,intranasal or oral inhalation, intranasal drops) routes ofadministration, depending on the agent and disease or condition to betreated. Administration can be local or systemic as indicated. Thepreferred mode of administration can vary depending upon the agentchosen, and the condition (e.g., disease) being treated, however, oralor parenteral administration is generally preferred.

The antibody, antigen-binding fragment thereof, immunoglobulin heavychain, antigen-binding portion thereof, immunoglobulin light chain,antigen-binding portion thereof, fusion protein and/or immuno-conjugateand any other therapeutic agent to be administered can be administeredas a neutral compound or as a salt. Salts of compounds (e.g., anantibody) containing an amine or other basic group can be obtained, forexample, by reacting with a suitable organic or inorganic acid, such ashydrogen chloride, hydrogen bromide, acetic acid, perchloric acid andthe like. Compounds with a quaternary ammonium group also contain acounteranion such as chloride, bromide, iodide, acetate, perchlorate andthe like. Salts of compounds containing a carboxylic acid or otheracidic functional group can be prepared by reacting with a suitablebase, for example, a hydroxide base. Salts of acidic functional groupscontain a countercation such as sodium, potassium and the like.

The antibody, antigen-binding fragment thereof, immunoglobulin heavychain, antigen-binding portion thereof, immunoglobulin light chain,antigen-binding portion thereof, fusion protein and/or immuno-conjugatecan be administered to the individual as part of a pharmaceuticalcomposition, for example, for modulation (e.g., inhibition) of CXCR3function (e.g., ligand binding and/or signal transduction, intracellularcalcium release, chemotaxis, cell differentiation, cell proliferation orcell activation), or treating a subject having a disease describedherein. The pharmaceutical composition can comprise an antibody,antigen-binding fragment, fusion protein and/or immuno-conjugate of theinvention and a pharmaceutically or physiologically acceptable carrier.In another embodiment, the pharmaceutical composition can comprise animmuglobulin heavy chain or antigen-binding portion thereof and aphysiologically acceptable carrier. The foregoing composition canfurther comprise a complementary light chain or antigen-binding portionthereof. In another embodiment, the pharmaceutical composition cancomprise an immunoglobulin light chain or antigen-binding portionthereof and a physiologically acceptable carrier. The foregoingcomposition can further comprise a complementary heavy chain orantigen-binding portion thereof. Formulation will vary according to theroute of administration selected (e.g., solution, emulsion, capsule).Suitable pharmaceutical and physiological carriers can contain inertingredients which do not interact with the antibody, fusion proteinand/or immuno-conjugate. Standard pharmaceutical formulation techniquescan be employed, such as those described in Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. Suitable pharmaceuticalcarriers for parenteral administration include, for example, sterilewater, physiological saline, bacteriostatic saline (saline containingabout 0.9% benzyl alcohol), phosphate-buffered saline, Hank's solution,Ringer's-lactate and the like. Methods for encapsulating compositions(such as in a coating of hard gelatin or cyclodextran) are known in theart (Baker, et al., “Controlled Release of Biological Active Agents”,John Wiley and Sons, 1986). For inhalation, the agent can be solubilizedand loaded into a suitable dispenser for administration (e.g., anatomizer, nebulizer or pressurized aerosol dispenser).

Furthermore, the antibody, antigen-binding fragment thereof,immunoglobulin heavy chain, antigen-binding portion thereof,immunoglobulin light chain, antigen-binding portion thereof, or fusionprotein of the invention and other therapeutic agents that are proteinscan be administered via in vivo expression of the recombinant protein.In vivo expression can be accomplished via somatic cell expressionaccording to suitable methods (see, e.g. U.S. Pat. No. 5,399,346). Inthis embodiment, a nucleic acid encoding the protein can be incorporatedinto a retroviral, adenoviral or other suitable vector (preferably, areplication deficient infectious vector) for delivery, or can beintroduced into a transfected or transformed host cell capable ofexpressing the protein for delivery. In the latter embodiment, the cellscan be implanted (alone or in a barrier device), injected or otherwiseintroduced in an amount effective to express the protein in atherapeutically effective amount.

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way.

Examples Example 1 Generation of Human Antibodies which Bind Human CXCR3

XENOMOUSE® Technology mice (genetically engineered mice with humanantibody genes from Abgenix Inc., Fremont, Calif.) were immunized withtransfected L1.2 (murine B lymphoma cell line) cells which expressedhigh levels of CXCR3 receptor (CXCR3 transfectants). The production ofthese cells was described in U.S. Pat. No. 6,184,358 B1, the contents ofwhich are incorporated herein by reference in their entirety. The micewere immunized multiple times with CXCR3 transfectants using theprotocol presented Table 3.

TABLE 3 Immunization Protocol Time Day 1, Week 0 IP, mito Week 2 IP,mito Week 4 IP, mito Week 7.5 IP, mito Week 9 IP Week 11 IP, CFA Week 13IP, CFA Week 15 IP Week 30.5 IP Week 32.5 IV IP, mito = intraperitonealinjection of 0.2-0.3 ml phosphate-buffered saline (PBS) containing 1 ×10⁷ CXCR3 transfectants which had been treated with 25 μg/ml mitomycin Cfor 30 min IP = intraperitoneal injection of 0.2-0.3 ml PBS containing 1× 10⁷ CXCR3 transfectants CFA = Complete Freund's Adjuvant included ininjection IV = intravascular injection of 0.33 × 10⁷ CXCR3 transfectantsin 0.1 ml PBS

Three days after the last immunization, five animals were sacrificed,their spleen cells were isolated, combined and used to producehybridomas by standard methods. About 3×10⁸ spleen cells were fused with5- to 10-fold fewer SP2/0-Ag14 fusion partner cells (ATCC, No. CRL-1581,Manassas, Va.) or P3X63Ag8.653 fusion partner cells (ATCC, No. CRL-1580,Manassas, Va.), by brief resuspension in polyethylene glycol 1500 (50%in 75 mM HEPES, Boehringer-Mannheim, now Roche Applied Science,Indianapolis, Ind.), to generate the hybridomas. After fusion, the cellswere selected by incubation for two weeks in hypoxanthine, aminopterin,thymidine (HAT) medium specificity was assessed by staining CXCR3transfectants, control L1.2 cells and L1.2 cells that expressed humanCCR1, CCR2, CCR3, CCR4, CCR5, CXCR1, CXCR2, or CXCR4 (Qin et al. (1998)J Clin. Invest. 101:746-54). The binding of antibody from the hybridomasupernatants to receptor transfectants was detected byphycoerythrin-conjugated anti-human IgG with analysis by flow cytometry.Positive wells were those that contained supernatant which stained theCXCR3 transfectants, but not L1.2 cells which expressed the otherchemokine receptors tested. Positive wells were subcloned to yieldhybridoma cell lines. Hybridoma cultures were maintained in DMEM with10% fetal calf serum, 10 units/ml interleukin-6, 0.1% L-glutamine, and0.1% penicillin/streptomycin. Isotype determination was performed usingsecondary antibodies purchased from The Binding Site (Birmingham, UK)and revealed that both human mAb 5H7 and human mAb 7H5 are of the IgG2,kappa isotype.

Example 2 Characterization of Antibody Binding

The cell-type specificities and affinities of human mAb 5H7 and humanmAb 7H5 antibodies were characterized. For the studies described in thefollowing paragraphs, the human mAb 5H7 or human mAb 7H5 antibodies werebiotinylated for detection by phycoerythrin conjugated to streptavidin(streptavidin-PE). The lymphocyte populations from normal donors werediscriminated by gating with forward and side scatters. The mouseanti-CXCR3 mAb 1C6 was used as a positive control.

The results revealed that a subpopulation of lymphocytes, but notmonocytes or granulocytes, were detected by human mAb 5H7. In a studyusing Cy5-conjugated anti-CD3, FITC-conjugated anti-CD4 and anti-CD8 tostain total T cells or subsets of T cells, respectively, and CD19 toidentify B cells, the staining pattern of human mAb 5H7 was similar tothat of murine mAb 1C6 (Qin et al. (1998) J. Clin. Invest. 101:746-54).

In another study, normal blood lymphocytes were cultured in the presenceof IL-2 (20 U/ml) for 14 days to activate T cells. These activated Tcells were incubated with 2 μg/ml biotinylated human mAb 5H7 followed bystreptavidin-PE. All the activated T cells were positively stained byhuman mAb 5H7, which is similar to the staining pattern of the mouseanti-human CXCR3 mAb 1C6.

The strength of human monoclonal antibody 5H7 and human monoclonalantibody 7H5 binding to CXCR3 was measured using CXCR3 transfectants.The mean streptavidin-PE fluorescence detected when 10 μg/ml of eachpurified mAb was used to stain CXCR3 transfectants was used as 100%fluorescence. The mean fluorescence intensity detected at severalantibody concentrations (indicated in FIG. 1) were plotted, and theconcentration of antibody which produced 50% fluorescence (EC50) wascalculated using KALEIDAGRAPH™ data analysis software (Synergy Software,Reading, Pa.). The calculated EC50 values are 0.5 nM, 1.2 nM and 2.3 nMfor human mAb 5H7, human mAb 7H5 and mouse mAb 1C6, respectively (FIG.1).

Example 3 Human Monoclonal Antibodies 5H7 and 7H5 Inhibit CXCR3-MediatedChemotaxis

Inhibition of CXCR3-mediated chemotaxis by human mAb 5H7 and human mAb7H5 was studied in an in vitro chemotaxis assay and compared to theinhibition by mouse mAb 1C6. CXCR3/L1.2 transfectants were used in thechemotaxis assays. Chemokines IP-10, MIG and I-TAC (PeproTech, Inc,Rocky Hill, N.J.) were diluted in RPMI/0.5% BSA to 5 nM in the bottomchamber of TRANSWELL® cell culture insert plates (Corning IncorporatedLife Sciences, Acton, Mass.). About 1×10⁶ transfectants were added tothe top chamber without or with 100 μg/ml of purified antibody, and theplates were incubated at 37° C., 5% CO₂ for 4 hours. Cells whichMigrated to the lower chamber were collected, suspended, and counted byflow cytometry for a set time of 30 seconds.

The results of chemotaxis assays using purified 5H7, 1C6 or 7H5antibodies are presented in Table 4 and show that each antibodyinhibited chemotaxis.

TABLE 4 Number of Cells Which Migrated to the Bottom Chamber in aChemotaxis Assay Ligand in Bottom Chamber Top Chamber IP-10 MIG I-TACControl, no antibodies 4151 3402 11550 1C6 51 2346 7664 5H7, subclone 11235 1268 2676 5H7, subclone 2 486 836 1970 7H5, subclone 2 1252 13072526 7H5, subclone 4 2760 2197 7495 No ligand in bottom 0 0 0

FIG. 2 shows the results of another study demonstrating thatCXCR3-mediated chemotaxis induced by three ligands of CXCR3 (IP10, MIGand I-TAC) was inhibited by high amounts of purified antibodies from twosubclones of human 5H7 hybridoma (subclones 2-4 and 2-6, fractions (1)and (2) of each) and from mouse 1C6 hybridoma.

The abilities of human mAb 5H7 and murine mAb 1C6 to inhibitCXCR3-mediated chemotaxis induced by IP-10, MIG or I-TAC werequantified. Maximum chemotaxis was the number of cells which Migrated tothe lower chamber (which contained IP-10, MIG or I-TAC) in the absenceof any antibodies. Percent inhibition was calculated as follows:100×[1−(s−b)/(t−b)], where s is the number of cells which Migrated tothe lower chamber in test wells, b is the background Migration in theabsence of chemokine, and t is the maximum chemotaxis. Differentconcentrations of antibody were included with CXCR3 transfectants in thetop chamber and chamotaxis was assessed. The concentration of antibodywhich inhibited chemotaxis by 50% (IC50) was calculated usingKALEIDAGRAPH™ data analysis software (Synergy Software, Reading, Pa.).The inhibition profile for human mAb 5H7 is shown in FIG. 3 and for 1C6is shown in FIG. 4. The IC50 of human mAb 5H7 against IP-10 is 0.7μg/ml, against MIG is 2.9 μg/ml and against I-TAC is 1.2 μg/ml. The IC50of murine mAb 1C6 against IP-10 is 0.8 μg/ml, against MIG is 93 μg/mland against I-TAC is 24 μg/ml. At 50 μg/ml. 1C6 inhibited about 40% ofMIG- and 60% of I-TAC-induced CXCR3-medicated chemotaxis, respectively.

Example 4 Human mAbs 5H7 and 7H5 are Not CXCR3 Agonists

Human T cells were activated by anti-CD3 mAb (OKT3, 3 μg/ml) for threedays. Then they were expanded in the presence of IL-2 (2 pg/ml) for upto 4 weeks. Cells were loaded with Fluo-3 (Molecular Probes, Inc.,Eugene, Oreg.) at 10 nM for 30 minutes at 37° C., washed once in buffer(Hank's Balanced Salt Solution supplemented with 0.1% BSA). The cellswere resuspended in the buffer and stimulated with IP10, MIG, or I-TACin the presence of purified human mAb 5H7, purified human mAb 7H5 orpurified murine mAb 1C6. The changes in real time were recorded withFACS flow cytometry measuring fluorescence intensity of FL 1 vs. time.None of the mAbs tested elicited a noticeable intracellular Ca++mobilization at 100 μg/ml of antibody concentration. The CXCR3 ligandI-TAC was used at 5 nM as the positive control to demonstrateintracellular Ca++ mobilization induced by chemokines. Other ligands,IP-10 and MIG induced similar responses at this concentration.

Example 5 Determination of the Primary Structure of Human mAb 5H7 andHuman mAb 7H5

RNA was isolated from multiple subclones of 5H7 and 7H5 hybridomas.Transcribed heavy and light chain variable regions of hybridomaimmunoglobulin genes were amplified by polymerase chain reaction (PCR)using an IG-PRIMER Set (immunoglobulin amplification reagents, Novagen,Inc., Madison, Wis.) having degenerate primers. A minilibrary wasprepared from the amplified variable region cDNAs using the TOPO®Cloning kit (Invitrogen Life Technologies, Carlsbad, Calif.). All theminilibrary clones were sequenced to identify the sequences encoding thehybridoma heavy and light chains. Through aligning overlapping segments,the sequence of one active heavy chain and one active light chain wasdetermined for each hybridoma.

Further confirmation of the human mAb 5H7 structure and function wasachieved by inserting the sequences into an expression vector fortransfection of non-hybridoma cells. Nucleic acid sequence encoding thevariable regions (VH and VK) of human mAb 5H7 (SEQ ID NOs:1 and 9) wereinserted in the pLKTOK59 vector. This vector can be used to express anantibody (heavy and light chains expression driven by the EF-1apromoter) that contains a human kappa constant region, and a human IgG1constant region that contains mutations at positions L235A and G237A(IgG1-FcRmut). These mutations inhibit the binding of the constantregion to human Fc receptors and inhibit the initiation ofantibody-dependent cellular cytotoxicity reactions. The nucleic acidencoding the variable region of the immunoglobulin heavy chain (VH) of5H7 and containing SEQ ID NO:1 was prepared by PCR using the followingprimers: TTACCCAATTGTGTCCTGTCCCAGGTGCAGGTGGTGCAGTCTGGGGCTG (SEQ IDNO:38) and TGGAGGCTGAGCTGACGGTGACCGTGGTCCCTTGGCCCCAGACGTCCATA (SEQ IDNO:39). These contain restriction sites (an Mfe I restriction site inthe leader sequence and the Blp I restriction site in the 3′ end) whichwere used for in-frame insertion of the human mAb 5H7 VH nucleic acidsequence after the leader sequence of the heavy chain construct in thepLKTOK59D vector. The nucleic acid encoding the variable region of theimmunoglobulin light chain (VK) of 5H7 and containing SEQ ID NO:9 wasprepared by PCR using the following primers:TTCCCAGGGTCCCGTTCCGACATCCAGATGACCCAGTCTCCATCCTCCCTG (SEQ ID NO:40),AGCCACCGTACGCTTAATCTCCAGTCGTGTCCCTTG (SEQ ID NO:41),CTGAACCTTGATGGGACTCCACTTTGCAAACTGGATGCGCCATAGAT (SEQ ID NO:42),TCCAGTTTGCAAAGTGGAGTCCCATCAAGGTTCAGTGGCAGTGGATCTG (SEQ ID NO:43), andAGCCACCGTACGCTTAATCTCCAGTCGTGTCCCTTGGCCGAAACTGATAGGGACTCTGAAACTCTGTTGACAGTAGTAAGTTG (SEQ ID NO:44). The first two and the last(SEQ ID NOs:40, 41 and 44) contain restriction sites (a PpuM Irestriction site in the leader sequence and the Bs1W I restriction sitein the 3′ end) which were used for in-frame insertion of the human mAb5H7 VK nucleic acid sequence after the leader sequence of the lightchain construct in the pLKTOK59D vector. The resulting vector, namedpLKTOK59D-5H7VHVK was transfected into 293T (transformed human renalepithelial line expressing two viral oncogenes, adenovirus Ela and SV40large T antigen) cells. Conditioned culture medium was collected fromthese transient transfectants.

All documents cited throughout this application including references,pending patent applications and published patents, are hereby expresslyincorporated herein by reference in their entirety.

Although preferred embodiments of the invention have been describedusing specific terms, such description are for illustrative purposesonly, and it is to be understood that changes and variations may be madewithout departing from the spirit or scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. cm What is claimedis:

1. A method for treating a subject having a disorder selected from thegroup consisting of an inflammatory disorder, an autoimmune disease, anallograft rejection and cancer, comprising administering to said subjectan effective amount of an antibody or antigen-binding fragment thereof,wherein the antibody or antigen-binding fragment thereof binds a CXCR3protein, and a) said antibody or antigen-binding fragment thereofcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2 and HCDR3) comprising the amino acid sequences: HCDR1: SEQ ID NO:4; HCDR2: SEQ ID NO: 6; and HCDR3: SEQ ID NO: 8; and  three light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3) comprisingthe amino acid sequences: LCDR1: SEQ ID NO: 12; LCDR2: SEQ ID NO: 14;and LCDR3: SEQ ID NO: 16; or b) said antibody or antigen-bindingfragment thereof comprises three heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3) comprising the amino acid sequences:HCDR1: SEQ ID NO: 20; HCDR2: SEQ ID NO: 22; and HCDR3: SEQ ID NO: 24;and  three light chain complementarity determining regions (LCDR1, LCDR2and LCDR3) comprising an amino acid sequences: LCDR1: SEQ ID NO: 28;LCDR2: SEQ ID NO: 30; and LCDR3: SEQ ID NO:
 32. 2. The method of claim1, wherein the antibody or antigen-binding fragment thereof: inhibitsbinding of a ligand to said CXCR3 protein; inhibits a function of aCXCR3 protein selected from the group consisting of CXCR3 ligand-inducedintracellular calcium release, CXCR3 ligand-induced chemotaxis, CXCR3ligand-induced cell differentiation, CXCR3 ligand-induced cellproliferation and CXCR3 ligand-induced cell activation; competitivelyinhibits binding of human monoclonal antibody 5H7 or human monoclonalantibody 7H5 to said CXCR3 protein; or has the epitopic specificity ofhuman monoclonal antibody 5H7 or human monoclonal antibody 7H5.
 3. Themethod of claim 2, wherein said CXCR3 ligand is selected from the groupconsisting of IP-10, MIG and I-TAC.
 4. The method of claim 1, whereinsaid antibody or antigen-binding fragment thereof is selected from thegroup consisting of a human antibody, an antigen-binding fragment of ahuman antibody, a humanized antibody, an antigen-binding fragment of ahumanized antibody, a chimeric antibody and an antigen-binding fragmentof a chimeric antibody.
 5. The method of claim 1, wherein saidantigen-binding fragment is selected from the group consisting of an Fabfragment, an Fab′ fragment, an F(ab′)₂ fragment and an Fv fragment. 6.The method of claim 1, wherein the antibody or antigen-binding fragmentthereof comprises a) a heavy chain variable region encoded by SEQ IDNO:1 and a light chain variable region encoded by SEQ ID NO:9, or b) aheavy chain variable region encoded by SEQ ID NO:17, and a light chainvariable region encoded by SEQ ID NO:25.
 7. The method of claim 1,wherein the antibody or antigen-binding fragment thereof comprises a) aheavy chain variable region comprising SEQ ID NO:2 and a light chainvariable region comprising SEQ ID NO:10, or b) a heavy chain variableregion comprising SEQ ID NO:18, and a light chain variable regioncomprising SEQ ID NO:26.
 8. The method of claim 1, wherein the antibodyor antigen-binding portion thereof is an immunoconjugate.
 9. The methodof claim 8, wherein said immunoconjugate comprises a therapeutic agent.10. The method of claim 1, wherein the inflammatory disease isulcerative colitis.
 11. The method of claim 1, wherein the inflammatorydisease is graft rejection.
 12. The method of claim 1, wherein theinflammatory disease is a respiratory inflammatory disease.
 13. Themethod of claim 1, wherein the autoimmune disease is multiple sclerosis.14. The method of claim 1, wherein the autoimmune disease is rheumatoidarthritis.
 15. A method for treating a subject having a disorderselected from the group consisting of an inflammatory disorder, anautoimmune disease, an allograft rejection and cancer an inflammatorydisorder, comprising administering to said subject an effective amountof an antibody or antigen-binding fragment of an antibody selected fromthe group consisting of the antibody produced by hybridoma 5H7 (ATCC®Accession No. PTA-5388) and the antibody produced by hybridoma 7H5(ATCC® Accession No. PTA-5389).
 16. The method of claim 1, wherein theheavy chain of the antibody comprises the immunoglobulin heavy chain oran antigen-binding portion thereof of the antibody produced by thehybridoma 5H7(ATCC® Accession No. PTA-5388) or of the antibody producedby hybridoma 7H5 (ATCC® Accession No. PTA-5389).
 17. The method of claim1, wherein the light chain of the antibody comprises the immunoglobulinlight chain or an antigen-binding portion thereof of the antibodyproduced by the hybridoma 5H7 (ATCC® Accession No. PTA-5388) or of theantibody produced by hybridoma 7H5 (ATCC® Accession No. PTA-5389). 18.An isolated cell which produces an antibody or antigen-binding fragment,wherein the antibody or antigen-binding fragment thereof binds a CXCR3protein, and a) said antibody or antigen-binding fragment thereofcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2 and HCDR3) comprising the amino acid sequences: HCDR1: SEQ ID NO:4; HCDR2: SEQ ID NO: 6; and HCDR3: SEQ ID NO: 8; and  three light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3) comprisingthe amino acid sequences: LCDR1: SEQ ID NO: 12; LCDR2: SEQ ID NO: 14;and LCDR3: SEQ ID NO: 16; or b) said antibody or antigen-bindingfragment thereof comprises three heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3) comprising the amino acid sequences:HCDR1: SEQ ID NO: 20; HCDR2: SEQ ID NO: 22; and HCDR3: SEQ ID NO: 24;and  three light chain complementarity determining regions (LCDR1, LCDR2and LCDR3) comprising an amino acid sequences: LCDR1: SEQ ID NO: 28;LCDR2: SEQ ID NO: 30; and LCDR3: SEQ ID NO:
 32. 19. The isolated cell ofclaim 18, wherein the antibody or antigen-binding fragment thereofcomprises a) a heavy chain variable region encoded by SEQ ID NO:1 and alight chain variable region encoded by SEQ ID NO:9, or b) a heavy chainvariable region encoded by SEQ ID NO:17, and a light chain variableregion encoded by SEQ ID NO:25.
 20. The isolated cell of claim 18,wherein the antibody or antigen-binding fragment thereof comprises a) aheavy chain variable region comprising SEQ ID NO:2 and a light chainvariable region comprising SEQ ID NO:10, or b) a heavy chain variableregion comprising SEQ ID NO:18, and a light chain variable regioncomprising SEQ ID NO:26.
 21. An isolated and/or recombinant nucleic acidcomprising a nucleotide sequence that encodes an immunoglobulin heavychain or antigen-binding portion thereof comprising three heavy chaincomplementarity determining regions (HCDR1, HCDR2 and HCDR3), whereinHCDR3 comprises an amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 8, the amino acidsequence of SEQ ID NO: 8 wherein one, two or three amino acid residuesare conservatively substituted, the amino acid sequence of SEQ ID NO:24and the amino acid sequence of SEQ ID NO: 24 wherein one, two or threeamino acid residues are conservatively substituted, and wherein anantibody comprising said heavy chain or antigen-binding portion thereofand a complementary light chain or antigen-binding portion of acomplementary light chain binds a CXCR3 protein.
 22. An expressionconstruct comprising a recombinant nucleic acid that encodes animmunoglobulin heavy chain or antigen-binding portion thereof comprisingthree heavy chain complementarity determining regions (HCDR1, HCDR2 andHCDR3), wherein HCDR3 comprises an amino acid sequence selected from thegroup consisting of the amino acid sequence of SEQ ID NO: 8, the aminoacid sequence of SEQ ID NO: 8 wherein one, two or three amino acidresidues are conservatively substituted, the amino acid sequence of SEQID NO:24 and the amino acid sequence of SEQ ID NO: 24 wherein one, twoor three amino acid residues are conservatively substituted, and whereinan antibody comprising said heavy chain or antigen-binding portionthereof and a complementary light chain or antigen-binding portion of acomplementary light chain binds a CXCR3 protein.
 23. A host cellcomprising a recombinant nucleic acid that encodes an immunoglobulinheavy chain or antigen-binding portion thereof comprising three heavychain complementarity determining regions (HCDR1, HCDR2 and HCDR3),wherein HCDR3 comprises an amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 8, the amino acidsequence of SEQ ID NO: 8 wherein one, two or three amino acid residuesare conservatively substituted, the amino acid sequence of SEQ ID NO:24and the amino acid sequence of SEQ ID NO: 24 wherein one, two or threeamino acid residues are conservatively substituted, and wherein anantibody comprising said heavy chain or antigen-binding portion thereofand a complementary light chain or antigen-binding portion of acomplementary light chain binds a CXCR3 protein.
 24. An isolated cellthat produces an immunoglobulin heavy chain or antigen-binding portionthereof comprising three heavy chain complementarity determining regions(HCDR1, HCDR2 and HCDR3), wherein HCDR3 comprises an amino acid sequenceselected from the group consisting of the amino acid sequence of SEQ IDNO: 8, the amino acid sequence of SEQ ID NO: 8 wherein one, two or threeamino acid residues are conservatively substituted, the amino acidsequence of SEQ ID NO:24 and the amino acid sequence of SEQ ID NO: 24wherein one, two or three amino acid residues are conservativelysubstituted, and wherein an antibody comprising said heavy chain orantigen-binding portion thereof and a complementary light chain orantigen-binding portion of a complementary light chain binds a CXCR3protein.
 25. An isolated and/or recombinant nucleic acid comprising anucleotide sequence that encodes an immunoglobulin light chain orantigen-binding portion thereof comprising three light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3), whereinLCDR3 comprises an amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 16, the amino acidsequence of SEQ ID NO: 16 wherein one or two amino acid residues areconservatively substituted, the amino acid sequence of SEQ ID NO:32 andthe amino acid sequence of SEQ ID NO: 32 wherein one or two amino acidresidues are conservatively substituted, and wherein an antibodycomprising said light chain or antigen-binding portion thereof and acomplementary heavy chain or antigen-binding portion of a complementarylight chain binds a CXCR3 protein.
 26. An expression constructcomprising a recombinant nucleic acid that encodes an immunoglobulinlight chain or antigen-binding portion thereof comprising three lightchain complementarity determining regions (LCDR1, LCDR2 and LCDR3),wherein LCDR3 comprises an amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 16, the amino acidsequence of SEQ ID NO: 16 wherein one or two amino acid residues areconservatively substituted, the amino acid sequence of SEQ ID NO:32 andthe amino acid sequence of SEQ ID NO: 32 wherein one or two amino acidresidues are conservatively substituted, and wherein an antibodycomprising said light chain or antigen-binding portion thereof and acomplementary heavy chain or antigen-binding portion of a complementarylight chain binds a CXCR3 protein.
 27. A host cell comprising arecombinant nucleic acid that encodes an immunoglobulin light chain orantigen-binding portion thereof comprising three light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3), whereinLCDR3 comprises an amino acid sequence selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 16, the amino acidsequence of SEQ ID NO: 16 wherein one or two amino acid residues areconservatively substituted, the amino acid sequence of SEQ ID NO:32 andthe amino acid sequence of SEQ ID NO: 32 wherein one or two amino acidresidues are conservatively substituted, and wherein an antibodycomprising said light chain or antigen-binding portion thereof and acomplementary heavy chain or antigen-binding portion of a complementarylight chain binds a CXCR3 protein.
 28. An isolated cell that produces animmunoglobulin light chain or antigen-binding portion thereof comprisingthree light chain complementarity determining regions (LCDR1, LCDR2 andLCDR3), wherein LCDR3 comprises an amino acid sequence selected from thegroup consisting of the amino acid sequence of SEQ ID NO: 16, the aminoacid sequence of SEQ ID NO: 16 wherein one or two amino acid residuesare conservatively substituted, the amino acid sequence of SEQ ID NO:32and the amino acid sequence of SEQ ID NO: 32 wherein one or two aminoacid residues are conservatively substituted, and wherein an antibodycomprising said light chain or antigen-binding portion thereof and acomplementary heavy chain or antigen-binding portion of a complementarylight chain binds a CXCR3 protein.